Lab 2. Isolation of mononuclear cells from peripheral blood and separation into subpopulations

Save this PDF as:
 WORD  PNG  TXT  JPG

Size: px
Start display at page:

Download "Lab 2. Isolation of mononuclear cells from peripheral blood and separation into subpopulations"

Transcription

1 Lab 2 Isolation of mononuclear cells from peripheral blood and separation into subpopulations Supervisors: Sissela Broos tel: Niclas Olsson tel:

2 Aim: To gain an insight into the purification of lymphocytes and analysis of antigen expression on different cell populations. Flowchart Filter material Ficoll-paque Lymphocytes and monocytes (PBMC) Binding to plastics Rosetting Adherent cells (monocytes) T-cells fraction B-cell fraction (incl. monocytes) 2

3 Introduction Blood contain different types of cells, for example red blood cells (erythrocytes), which transport oxygen to all tissues in the body and white blood cells (leukocytes), which are part of the immune system. In this lab we are going to look more closely at some of the white blood cell populations. We will do so using flow cytometry which is a common technique for analysis of cells in clinical laboratories. In order to perform this analysis, we must first separate different types of leukocytes from each other by using the unique characteristics of the cells. We will isolate T-lymphocytes, monocytes and B-lymphocytes. The aim of the lab is to give an insight into different cell separation methods, as well as how to analyze cells using flow cytometry. We will use a so called buffy coat, which is the remaining part when red blood cells are separated from the blood plasma in human blood. The buffy is obtained from the blood bank at Lund University Hospital. During day 1, we will carry out inital preparations to separate leukocytes from blood and subsequently isolate the different cell populations (monocytes, B-cells and T-cells). First, we will dispose off the red blood cells by performing a density gradient centrifugation on Ficoll-Paque. The PBMCs (peripheral blood mononuclear cells) will form a layer in the tube that will be further purified. Monocytes will be isolated by utilizing their ability to bind to plastic surfaces. T- lymphocytes will be separated using activated erythrocytes from sheep, which bind to a molecule on the surface of T-cells. The T-cell depleted fraction will then contain both B-cells and monocytes. The different cell populations will finally be studied using flow cytometry on day 2. All donors are tested before becoming approved blood donors, but there is still a risk that the material may be contagious when we receive it, so it is vital to work very carefully. Important to also bear in mind when working with cells: 1. Always keep the cell suspensions in tubes on ice. 2. Resuspend a cell pellet in a small volume of liquid first and then add more liquid. Otherwise the cells will form clumps. 3. Keep the lymphocytes at the right ph (6,8-7,4), even though they endure low ph better than high ph. 4. Work gently with the cells, they are small living creatures. Do not pipette quickly. 3

4 Isolation of mononuclear leukocytes In blood transfusions it is important that the blood does not contain any white blood cells. These are separated from the blood by filtration and the collected cells are referred to as filter material (formerly known as buffy coat, which is separated by centrifugation). From this you can obtain mononuclear cells using a density gradient centrifugation on Ficoll-Paque (density = 1.078). Differencies in density will separate lymphocytes from other blood cells. After the centrifugation three or four fractions are visible: Blood sample Ficoll-Paque Centrifugation Plasma Lymfoc., monocyt. & tromb. δ < Ficoll-Paque Granulocytes δ > Aggregated erythrocytes δ > Counting cells in a Bürker chamber 4

5 The proportion of viable cells can be determined by staining the cells with trypan blue. Viable cells will not become stained while dead cells will become blue. The cells have to be counted within 3-4 minutes or the viable cells will also take up the colour. Bürker chambers and cover slips should be properly cleaned in water and alcohol. The cover slip is attached to the glass by humidifying the borders of the glass. Dilute the cells in trypan blue and let the cell suspension slip in under the cover, into one of the chambers. Abundant cell suspension should be dried off using a Kleenex. Each field in the counting chamber is divided into 9 squares separated with triple lines and such a field (A-square) contain 0,1 µl. The A-square is further divided into 16 parts, B-squares (see figure below). When counting cells in an A-square, all cells that touch the right and the upper border should be included while cells on the left and bottom line should be excluded. Count at least a hundred cells (sometimes you may have to count more than one A-square) and use the average to determine the concentration of cells. Amount of viable cells/ml=viable cells in one A-square x 10 4 x dilution factor B-square A-square 5

6 Flow cytometry Flow cytometry is a mean of measuring certain physical and chemical characteristics of cells or particles as they travel in suspension one by one past a sensing point. The modern flow cytometer consists of a light source, collection optics, electronics and a computer to translate signals to data. In most modern cytometers the light source of choice is a laser, which emits coherent light at a specified wavelength. Scattered and emitted fluorescent light is collected by two lenses (one set in front of the light source and one set at right angles) and by a series of optics, beam splitters and filters to allow specific bands of fluorescence to be measured. Physical characteristics such as cell size, shape, internal complexity and any cell component or function that can be detected by a fluorescent compound can be examined. So the applications of Flow Cytometry are numerous, and this has led to the widespread use of these instruments in the biological and medical fields. The term "Flow Cytometry" derives from the measurement (meter) of single cells (cyto) as they flow past a series of detectors. The acronym FACS (Fluorescence Activated Cell Sorting) and Flow Cytometry are used interchangeably. The fundamental concept is that cells flow one at a time through a region of interrogation where multiple biophysical properties of each cell can be measured at rates of over 1000 cells per second. These biophysical properties are then correlated with biological and biochemical properties of interest. The high through-put of cells allows for rare cells, which may have inherent or inducible differences, to be easily detected and identified from the remainder of the cell population. 6

7 In order to make the measurement of biological/biochemical properties of interest easier, the cells are usually stained with fluorescent dyes, which bind specifically to cellular constituents. The dyes are excited by the laser beam, and emit light at a longer wavelength. Photo Multiplicator Tubes (PMT) detects the emitted light as an analogue electronic signal, which subsequently is converted to digital information to be analyzed in a computer. Three types of data are generated: Forward scatter (FSC) Approximate cell size Side/orthogonal scatter (SSC) Cell complexity or granularity Fluorescence Fluorescent labeling is used to investigate cell structure and function Scatter Forward and side scatter are used for preliminary identification of cells. In a peripheral blood sample, lymphocyte, monocyte and granulocyte populations can be defined on the basis of forward and side scatter. Forward and side scatter are also used to exclude debris and dead cells. Fluorescence Labeling cells with fluorescent dyes allows investigation of structure and function. Fluorescence intensities are typically measured at several different wavelengths simultaneously for each cell. Fluorescent probes are used to report the quantities of specific components of the cells. Fluorescent antibodies are often used to report the densities of specific surface receptors, and thus to distinguish subpopulations of differentiated cell types, including cells expressing a transgene. By making them fluorescent, the binding of viruses or hormones to surface receptors can be measured. Intracellular components can also be reported by fluorescent probes, including total DNA/cell (allowing cell cycle analysis), newly synthesized DNA, specific nucleotide sequences in DNA or mrna, filamentous actin, and any structure for which an antibody is available. Flow cytometry can also monitor rapid changes in intracellular free calcium, membrane potential, ph, or free fatty acids. Immunofluorescence, the most widely used application, involves the staining of cells with antibodies conjugated to fluorescent dyes such as fluorescein (FITC) and phycoerythrin (PE). This method is often used to label molecules on the cell surface, but antibodies can also be directed at intracellular targets in the cytoplasm. In direct staining the antibody is directly conjugated to a fluorescent dye (e.g. anti-cd4 PE). Cells are stained in one step. In indirect staining the primary antibody is not labeled. A second antibody conjugated to a fluorescent dye, and specific for the first antibody is added. For example, if the anti-cd4 antibody was a mouse IgG then the second antibody could be a rat antibody raised against mouse IgG. 7

8 Analysis The ability of Flow Cytometers to evaluate cells at an extremely rapid rate (e.g. up to 20,000 events per second) makes this technology ideally suited for the reliable and accurate quantitative analysis of selected physical properties of cells of interest. The sensitivity of these instruments for detecting the presence of molecules expressed at low levels is impressive; given high quality cell preparations and reagents, as few as 500 molecules per cell may be detected. At the Department of Immunotechnology the Becton-Dickinson FACS Canto II will be used for analysis. The BD FACSCanto system is built with blue (488 nm, air-cooled, 20 mw solid state) and red (633 nm, 17 mw HeNe) excitation sources. The laser beams are then routed via fiber optics to the beamshaping prisms where two lasers are projected onto separate spots in the flow cell. Here, the particles intercept with the laser excitation beams and optical signals are collected at the backside of the flow cell with a gel-coupled collection lens. The system has 6 different colour detectors and several different dyes can thereby be used. In addition the system has a very fast acquisition rates (up 10,000 events per sec). Cell sorting One of the properties of the larger flow cytometers is the ability to electronically deflect cells with preset, defined properties into a separate collection tube. In these instruments the fluidics hydrodynamically focuses the cell stream to within an uncertainty of a small fraction of a cell diameter and break the stream into uniform-sized droplets to separate individual cells. The electronics quantitate the faint flashes of scattered and fluorescent light, and, under computer control, electrically charge droplets containing cells of interest so that they can be deflected into a separate test tube or culture wells. For cell purification, flow cytometry is especially well suited for applications requiring high purity. Because multiple fluorochromes (e.g. up to five distinct fluorescent probes reacting with different cell associated molecules) can be assessed simultaneously, cell sorting by flow cytometry can separate complex mixtures of cells on the basis of multiple marker expression. At the Department of Immunotechnology we use a Becton-Dickinson FACS Aria for cell sorting. This Aria is a 9 detectors instrument with 3 lasers and digital acquisition rates of up to 70,000 events/second. The bitmapped sorting capability of the Aria digital electronics allows the operator to set up sophisticated sort decisions using logical gates combined with the Boolean operators AND, OR, and NOT. The Aria can also be used for multicolor assays with too many colors (nine) for the other instrument. 8

9 Important! The laboratory manual is only a guide. Some changes may occur during the lab and these must be documented and described in the lab report. DAY 1: 1. Fill a T75:a with 250 ml PBS. Cut the end of the filter tubing that is least blood filled and connect it to the vacuum tubing. Cut the other side and place it in the T75 with PBS. Turn on the vacuum and wait until all PBS has gone through the filter. Turn the vacuum off. 2. Disconnect the bucket from the vacuum tubing and carefully put it in the LAF bench. Carefully swirl the bucket and remove the top. Transfer the blood solution into 6 x 50 ml Falcon tubes using a 50 ml stripette. Fill up the last tube with PBS to balance in the centrifuge. 3. Centrifuge the tubes at 1800 rpm for 5 min (room temp.) 4. Aspirate the supernatant and leave about 10 ml in each tube. Pool the solution into two of the tubes and add PBS for a total volume of 75 ml. 5. Place 15 ml of Ficoll Paque into three 50 ml tubes/group. 6. Carefully transfer 25 ml of the diluted filter material ONTO the Ficoll Paque so that they form two separate layers. 7. Centrifuge in room temperature at 1800 rpm for 20 min without breaks. 8. The leukocytes can now be collected at the interphase between the Ficoll Paque and the plasma (se figure at page 4). Transfer them carefully using a 10 ml pipette or a pasteur pipette into four new 50 ml tubes. Try to avoid aspirating the plasma and Ficoll Paque. 9. Dilute the cell suspensions with PBS up to 50 ml in each tube. 9

10 10. Centrifuge 1250 rpm for 10 min (room temperature). 11. Aspirate the supernatants and be careful not to lose the cells. Resuspend the pellet BEFORE PBS is added. 12. Resuspend the cells in 10 ml cold PBS and pool the cells into one tube. Always keep the cells on ice after this point. 13. Centrifuge the cells at 900 rpm for 10 min (4 C). The leukocytes will now form a pellet while the trombocytes will stay in the supernatant. 14. Carefully aspirate the supernatant (the pellet is loose!!!). Resuspend the cells in 10 ml PBS, take out a sample and count the cells (dilute 50 times before counting) (see page 5). Repeat the washing step (number 10) while you are counting. 15. Resuspend the cells in cell culture medium (R5) to a cell concentration of 10 x 10 6 cells/ml. 16. Take out 3 times 50µl samples and transfer them to 3 different 5 ml tubes ( FACS tubes ) (=0,5 x 10 6 cells). Mark them with A1, A2 and A3 and the name of the group and keep them in the fridge before the analysis on day 2. 10

11 Binding of adherent cells to plastic surfaces The cells (from step 12) that we have at this stage are lymphocytes and monocytes. Monocytes could be separated by plastic adhesion, since they adhere to plastic surfaces and lymphocytes do not. Take ~ 75 x 10 6 cells (<1/3 of all cells) to this experiment. The rest of the cells are used for rosette formation (see next page). 1. Take ~ 75 x 10 6 cells (from step 12) and add R5 to a total volume of 15 ml. Transfer to a T75 flask. 2. Look at the cells in microscope. Incubate the cells at 37 C for 2-4 hours. 3. After 2 hours, take out the flasks and look at the cells in microscope. The monocytes that have adhered to the plastic surface looks like flattened amoebas. If you can not see this yet, incubate the cells for longer period of time and check them every half hour. 4. When there is a distinct carpet of cells in the bottom of the flask, remove all the media and put it in the waste. 5. Wash away T and B cells by adding new R5 media (10 ml) and then aspirate it. Repeat once again. 6. Look at the flask in microscope, if there are still cells present that are not bound to the surface, repeat step 5 until no unbound cells can be detected. 7. After the last wash, add 15 ml R5 to the flask in which you have monocytes. The monocytes are incubated at 37 C over night. The cells will then no longer be bound to the surface and can be used in another test (in our case we will stain them with fluorescence-labeled anti-monocytes-antibodies and analyze them using flow cytometry). 11

12 Rosette formation of T-cells with red blood cells from sheep Erythrocytes from sheep (FRBK) have a natural affinity for human T lymphocytes (binds to a marker on T lymphocytes that is called CD2). This has for a long time been used for positive selection of T lymphocytes. When the surface of T lymphocytes are covered with erythrocytes small aggregates (rosettes) are formed that can easily be separated from the other lymphocytes by gradient centrifugation (Ficoll or Percoll). 1. The rest of the cellsuspension (~12.5 ml from step 15 above, 10*10 6 cells/ml) is divided into two 50 ml tubes. Resuspend a tube with FRBK (10%) by carefully tilting it upside down a couple of times. Add 10 ml of activated FRBK to each tube with cellsuspension. Keep Cold! Centrifuge 900 rpm for 3 min. Breaks off!! 2. Incubate on ice for 25 min. 3. Add 4 ml Ficoll (1.078 g/ml) to 4 different 15 ml tubes. Stop the incubation above. Dissolve the pellet and transfer the suspension really gently ONTO the Ficoll so that 2 separate layers will form. Leave a little droplet of blood in the tube. 4. Centrifuge at 4 C, 2000 rpm for 15 min. Breaks off!! 5. While the tubes are centrifuged put the droplet of blood that you saved on a slide. Look at the rosettes in microscope. Take a photo. 6. After centrifugation the T-lymphocytes are in the pellet while the non- T-fraction is in the interphase. Carefully suck up the interphase and put it in a 15 ml-tube. Dilute the non-t-fraction to 15 ml with R5 and wash (10 min, 1000 rpm). Resuspend in 2 ml and count the cells (dilute 100 times for the counting). After that, resuspend the cells to a density of 10*10 6 cells/ml. Take out samples of 0,5 x 10 6 cells (=50ul) and add to 3 tubes ( FACS tubes ). Mark the tubes!! (D1, D2 resp. D3) 7. Take away the rest of the supernatant (from step 6.). The pellet (T lymphocytes) could be quit loose, so be careful! Prepare a pipette for 500 µl with a new tip. Add 4 ml dh 2 O/tube, pipette once up and down and then add 500µl 10x NaCl (1,4 M) within 10 seconds! 12

13 8. Add R5 up to 15 ml in each tube and centrifuge 1250 rpm for 10 min. Pool the cell suspensions from the different tubes to a 50 ml-tube. 9. Wash the cells one more time in 10 ml R5. Resuspend the cells in 2 ml. Count the cells (diluted 100 times for the counting). Resuspend the cells to 10 x 10 6 /ml in R5. Count carefully! Save half a million of cells (50 µl) in 6 different 5 ml tubes ( FACS tubes ). Mark the tubes!! (C1, C2, C3, C4, resp. C5) 13

14 DAY 2: Binding of adherent cells to plastic surface 1. Take out the monocytes from the incubator. Check that the monocytes have come off from the plastic surface. If not, try to hit the flask gently. Transfer the cell suspension to a 50 ml tube and centrifuge. The pellet will be really small so be careful when you aspirate the suspension on the top. Resuspend the cells in 0,5ml and count (dilute 10 times). Dilute to a concentration of 10*10 6 cells/ml. Transfer 0,5 x 10 6 cells to three different 5 ml tubes (50 µl/tube) and mark the tubes with B1, B2 resp B3. Surface staining of fractionated PBMC 1. Add 1 µl mouse Ig blocking (100ul/ml) solution to each of the 14 samples. 2. The staining of the cell is performed in a total of 100 µl where 50 µl is cell suspension and 50 µl is an antibody cocktail. Add 50 µl of each prepared antibody cocktail with marked antibodies to the cell suspension as follow: Sample: Cocktail: A1, B1, C1, D1 unstained A2, B2, C2, D2 isotype-control A3, B3, C3, D3 CD3/CD19/CD14 C4 CD3/CD4/CD8 C5 CD4/ CD8/CD45RO 3. Incubate on ice for 30 min. 4. Wash 1 x 3 ml with PBS/1% BSA (5 min, 1250 rpm). 5. Resuspend the cells in 500 µl PBS/1%BSA. 6. Run FACS-analysis. 14

15 Staining with fluorescent labeled antibodies for flow cytometry analysis By using fluorescent labeled antibodies one can decide what type of cells and their internal relations in a sample. By sampling at different stages in the purification process, one can follow the purification. Each fraction (A, B, C, D) was stained with the following antibodies: Sample 1 (A, B, C, D): Sample 2 (A, B, C, D): Sample 3 (A, B, C, D): Unstained cells mouse IgG1 FITC mouse IgG1 PE (isotype control) α CD3-APC α CD19-FITC α CD14-PE Sample C4: α CD3 APC α CD4 PE α CD8 FITC Sample C5: α CD4 PE α CD8 FITC α CD45RO APC α CD19 stain B-cells α CD14 stain monocytes α CD3 stain T-cells (both CD4- and CD8-positive T-cells) α CD4 stain T-helper cells of type T H 1 and T H 2 α CD8 stain cytotoxic T-lymphocytes (CTLs) α CD45RO stain memory T-cells Which cell type is the dominating one in each fraction? A... B... C... D... 15

Introduction to flow cytometry

Introduction to flow cytometry Introduction to flow cytometry Flow cytometry is a popular laser-based technology. Discover more with our introduction to flow cytometry. Flow cytometry is now a widely used method for analyzing the expression

More information

Katharina Lückerath (AG Dr. Martin Zörnig) adapted from Dr. Jörg Hildmann BD Biosciences,Customer Service

Katharina Lückerath (AG Dr. Martin Zörnig) adapted from Dr. Jörg Hildmann BD Biosciences,Customer Service Introduction into Flow Cytometry Katharina Lückerath (AG Dr. Martin Zörnig) adapted from Dr. Jörg Hildmann BD Biosciences,Customer Service How does a FACS look like? FACSCalibur FACScan What is Flow Cytometry?

More information

Below is a list of things you should be aware of before you schedule your sort.

Below is a list of things you should be aware of before you schedule your sort. Sorting at the Flow Cytometry Facility At the present time, the assistance of a trained cell sorter operator is needed for all sorting applications. As such, if you are planning a first time sort you will

More information

These particles have something in common

These particles have something in common These particles have something in common Blood cells Chromosomes Algae Protozoa Certain parameters of these particles can be measured with a flow cytometer Which parameters can be measured? the relative

More information

Introduction to Flow Cytometry

Introduction to Flow Cytometry Introduction to Flow Cytometry presented by: Flow Cytometry y Core Facility Biomedical Instrumentation Center Uniformed Services University Topics Covered in this Lecture What is flow cytometry? Flow cytometer

More information

NPTEL Biotechnology Tissue Engineering. Cell separation

NPTEL Biotechnology Tissue Engineering. Cell separation Cell separation S. Swaminathan Director Centre for Nanotechnology & Advanced Biomaterials School of Chemical & Biotechnology SASTRA University Thanjavur 613 401 Tamil Nadu Joint Initiative of IITs and

More information

MEASURABLE PARAMETERS: Flow cytometers are capable of measuring a variety of cellular characteristics such as:

MEASURABLE PARAMETERS: Flow cytometers are capable of measuring a variety of cellular characteristics such as: INTRODUCTION Flow Cytometry involves the use of a beam of laser light projected through a liquid stream that contains cells, or other particles, which when struck by the focused light give out signals

More information

No-wash, no-lyse detection of leukocytes in human whole blood on the Attune NxT Flow Cytometer

No-wash, no-lyse detection of leukocytes in human whole blood on the Attune NxT Flow Cytometer APPLICATION NOTE Attune NxT Flow Cytometer No-wash, no-lyse detection of leukocytes in human whole blood on the Attune NxT Flow Cytometer Introduction Standard methods for isolating and detecting leukocytes

More information

Immunophenotyping peripheral blood cells

Immunophenotyping peripheral blood cells IMMUNOPHENOTYPING Attune Accoustic Focusing Cytometer Immunophenotyping peripheral blood cells A no-lyse, no-wash, no cell loss method for immunophenotyping nucleated peripheral blood cells using the Attune

More information

Standard Operating Procedure

Standard Operating Procedure 1.0 Purpose: 1.1 The characterisation of of main leukocyte subsets in peripheral blood cells from mice by flow cytometry. Reliable values of frequencies of leukocyte clusters are very much dependent on

More information

APPLICATION INFORMATION

APPLICATION INFORMATION DRAFT: Rev. D A-2045A APPLICATION INFORMATION Flow Cytometry 3-COLOR COMPENSATION Raquel Cabana,* Mark Cheetham, Jay Enten, Yong Song, Michael Thomas,* and Brendan S. Yee Beckman Coulter, Inc., Miami FL

More information

CFSE Cell Division Assay Kit

CFSE Cell Division Assay Kit CFSE Cell Division Assay Kit Item No. 10009853 Customer Service 800.364.9897 * Technical Support 888.526.5351 www.caymanchem.com TABLE OF CONTENTS GENERAL INFORMATION 3 Materials Supplied 4 Precautions

More information

Introduction to Flow Cytometry

Introduction to Flow Cytometry Outline Introduction to Flow Cytometry Basic Concept of Flow Cytometry Introduction to Instrument Subsystems Daisy Kuo Assistant Product Manager E-mail: daisy_kuo@bd.com BDBiosciences Application Examples

More information

PROTOCOL. Immunostaining for Flow Cytometry. Background. Materials and equipment required.

PROTOCOL. Immunostaining for Flow Cytometry. Background. Materials and equipment required. PROTOCOL Immunostaining for Flow Cytometry 1850 Millrace Drive, Suite 3A Eugene, Oregon 97403 Rev.0 Background The combination of single cell analysis using flow cytometry and the specificity of antibody-based

More information

Islet Viability Assessment by Single Cell Flow Cytometry

Islet Viability Assessment by Single Cell Flow Cytometry Islet Viability Assessment by Single Cell Flow Cytometry Page 1 of 8 Purpose: To comprehensively assess the viability of the islet cell preparation prior to transplantation. Tissue Samples: A sample containing

More information

RPCI 004 v.002 Staining Procedure For all Directly Conjugated Reagents (Whole Blood Method)

RPCI 004 v.002 Staining Procedure For all Directly Conjugated Reagents (Whole Blood Method) Immune Tolerance Network RPCI 004 v.002 Staining Procedure For all Directly Conjugated Reagents (Whole Blood Method) Author: Paul Wallace, Director, RPCI Laboratory of Flow Cytometry Approved by: Paul

More information

Chapter 6. Antigen-Antibody Properties 10/3/2012. Antigen-Antibody Interactions: Principles and Applications. Precipitin reactions

Chapter 6. Antigen-Antibody Properties 10/3/2012. Antigen-Antibody Interactions: Principles and Applications. Precipitin reactions Chapter 6 Antigen-Antibody Interactions: Principles and Applications Antigen-Antibody Properties You must remember antibody affinity (single) VS avidity (multiple) High affinity: bound tightly and longer!

More information

EdU Flow Cytometry Kit. User Manual

EdU Flow Cytometry Kit. User Manual User Manual Ordering information: (for detailed kit content see Table 2) EdU Flow Cytometry Kits for 50 assays: Product number EdU Used fluorescent dye BCK-FC488-50 10 mg 6-FAM Azide BCK-FC555-50 10 mg

More information

Flow Cytometry. What is Flow Cytometry? What Can a Flow Cytometer Tell Us About a Cell? Particle Size. Flow = Fluid Cyto = Cell Metry = Measurement

Flow Cytometry. What is Flow Cytometry? What Can a Flow Cytometer Tell Us About a Cell? Particle Size. Flow = Fluid Cyto = Cell Metry = Measurement What is Flow Cytometry? Flow Cytometry Basic Principle and Applications BD Biosciences Daisy Kuo (daisy_kuo@bd.com) Flow = Fluid Cyto = Cell Metry = Measurement A variety of measurements are made on cells,

More information

CyAn : 11 Parameter Desktop Flow Cytometer

CyAn : 11 Parameter Desktop Flow Cytometer CyAn : 11 Parameter Desktop Flow Cytometer Cyan ADP 3 excitation lines 488nm, 635nm, and UV or violet 11 simultaneous parameters FSC, SSC, and 7-9 colors with simultaneous width, peak, area, and log on

More information

WHOLE BLOOD LYSING SOLUTION FOR FLOW CYTOMETRIC APPLICATIONS

WHOLE BLOOD LYSING SOLUTION FOR FLOW CYTOMETRIC APPLICATIONS FOR IN-VITRO DIAGNOSTIC USE INVITROGEN CAL-LYSE TM Lysing Solution WHOLE BLOOD LYSING SOLUTION FOR FLOW CYTOMETRIC APPLICATIONS CAL-LYSE TM CATALOG No. GAS-010 250 tests 25 ml CATALOG No. GAS-010S-100

More information

CRITICAL ASPECTS OF STAINING FOR FLOW CYTOMETRY

CRITICAL ASPECTS OF STAINING FOR FLOW CYTOMETRY 页 码,1/6 CRITICAL ASPECTS OF STAINING FOR FLOW CYTOMETRY From Givan, A.L. (2000), chapter in In Living Color: Protocols in Flow Cytometry and Cell Sorting (R. Diamond and S. DeMaggio, eds). Springer, Berlin,

More information

Stepcount. Product Description: Closed transparent tubes with a metal screen, including a white matrix at the bottom. Cat. Reference: STP-25T

Stepcount. Product Description: Closed transparent tubes with a metal screen, including a white matrix at the bottom. Cat. Reference: STP-25T Product Description: Closed transparent tubes with a metal screen, including a white matrix at the bottom Cat. Reference: STP-25T Reagent provided:: 25 Stepcount tubes for 25 test INTENDED USE. Immunostep

More information

Single cell cloning of suspension cells with Calcein-AM and the Cellavista System

Single cell cloning of suspension cells with Calcein-AM and the Cellavista System Single cell cloning of suspension cells with Calcein-AM and the Cellavista System Introduction Single cell cloning (SCC) represents a critical step in cell line development for the production of biopharmaceuticals.

More information

Flow Cytometry A Basic Overview

Flow Cytometry A Basic Overview Flow Cytometry A Basic Overview Overview Flow cytometry is a powerful technology for investigating many aspects of cell biology and for isolating cells of interest. Flow cytometry utilizes highly focused,

More information

UNIVERSITY OF PÉCS MEDICAL SCHOOL FLOW CYTOMETRY AND CELL SEPARATION BIOPHYSICS 2. 2015 4th March Dr. Beáta Bugyi Department of Biophysics Flow cytometry and cell separation FLOW = STREAM OF FLUID in a

More information

INSIDE THE BLACK BOX

INSIDE THE BLACK BOX FLOW CYTOMETRY ESSENTIALS INSIDE THE BLACK BOX Alice L. Givan Englert Cell Analysis Laboratory of the Norris Cotton Cancer Center Dartmouth Medical School HOW NOT TO BE A FLOW CYTOMETRIST Drawing by Ben

More information

Let the BD FACSAria II accompany your best performance ever

Let the BD FACSAria II accompany your best performance ever Let the BD FACSAria II accompany your best performance ever Meet the new BD FACSAria II high-speed, fixed-alignment cuvette cell sorter. The first generation BD FACSAria system brought the complex world

More information

SAMPLE PREPARATION GUIDELINES FOR SORTS

SAMPLE PREPARATION GUIDELINES FOR SORTS The Flow Cytometry Facility SAMPLE PREPARATION GUIDELINES FOR SORTS Whatever your cell type - samples must be a high quality high viability single cell suspension. Cells with low cell viability will not

More information

DNA STAINING REAGENTS KIT STOCK NO. PI-STAIN

DNA STAINING REAGENTS KIT STOCK NO. PI-STAIN DNA STAINING REAGENTS KIT STOCK NO. PI-STAIN Table of Contents: Intended Use... 2 Background and Principle... 2 Reagents and Materials Provided... 2 Reagents and Materials that may be Required, but are

More information

Flow Cytometry for Everyone Else Susan McQuiston, J.D., MLS(ASCP), C.Cy.

Flow Cytometry for Everyone Else Susan McQuiston, J.D., MLS(ASCP), C.Cy. Flow Cytometry for Everyone Else Susan McQuiston, J.D., MLS(ASCP), C.Cy. At the end of this session, the participant will be able to: 1. Describe the components of a flow cytometer 2. Describe the gating

More information

Minimal residual disease detection in Acute Myeloid Leukaemia on a Becton Dickinson flow cytometer

Minimal residual disease detection in Acute Myeloid Leukaemia on a Becton Dickinson flow cytometer Minimal residual disease detection in Acute Myeloid Leukaemia on a Becton Dickinson flow cytometer Purpose This procedure gives instruction on minimal residual disease (MRD) detection in patients with

More information

ab133073 7-AAD/CFSE Cell- Mediated Cytotoxicity Assay Kit

ab133073 7-AAD/CFSE Cell- Mediated Cytotoxicity Assay Kit ab133073 7-AAD/CFSE Cell- Mediated Cytotoxicity Assay Kit Instructions for Use To quantify the cytotoxic effect of immune effector cells at a single cell level. This product is for research use only and

More information

ArC Amine Reactive Compensation Bead Kit

ArC Amine Reactive Compensation Bead Kit ArC Amine Reactive Compensation Bead Kit Catalog no. A1346 Table 1. Contents and storage information. Material Amount Composition Storage Stability ArC reactive beads (Component A) ArC negative beads (Component

More information

Isolation of Whole Mononuclear Cells from Peripheral Blood and Cord Blood

Isolation of Whole Mononuclear Cells from Peripheral Blood and Cord Blood PREPARATION OF HUMAN MONONUCLEAR CELL POPULATIONS AND SUBPOPULATIONS SECTION I This section describes procedures for the preparation of human mononuclear cell populations from peripheral blood (UNIT 7.1)

More information

Title: Mapping T cell epitopes in PCV2 capsid protein - NPB #08-159. Date Submitted: 12-11-09

Title: Mapping T cell epitopes in PCV2 capsid protein - NPB #08-159. Date Submitted: 12-11-09 Title: Mapping T cell epitopes in PCV2 capsid protein - NPB #08-159 Investigator: Institution: Carol Wyatt Kansas State University Date Submitted: 12-11-09 Industry summary: Effective circovirus vaccines

More information

ab139418 Propidium Iodide Flow Cytometry Kit for Cell Cycle Analysis

ab139418 Propidium Iodide Flow Cytometry Kit for Cell Cycle Analysis ab139418 Propidium Iodide Flow Cytometry Kit for Cell Cycle Analysis Instructions for Use To determine cell cycle status in tissue culture cell lines by measuring DNA content using a flow cytometer. This

More information

BACTERIAL ENUMERATION

BACTERIAL ENUMERATION BACTERIAL ENUMERATION In the study of microbiology, there are numerous occasions when it is necessary to either estimate or determine the number of bacterial cells in a broth culture or liquid medium.

More information

Principles of Flowcytometry

Principles of Flowcytometry Objectives Introduction to Cell Markers: Principles of Flowcytometry Michelle Petrasich NZIMLS Scientific Meeting August 24, 2010, Paihia What are cell markers How do we detect them Production of Monoclonal

More information

Boundary-breaking acoustic focusing cytometry

Boundary-breaking acoustic focusing cytometry Boundary-breaking acoustic focusing cytometry Introducing the Attune NxT Acoustic Focusing Cytometer a high-performance system that s flexible enough for any lab One of the main projects in my laboratory

More information

Application Note 10. Measurement of Cell Recovery. After Sorting with a Catcher-Tube-Based. Cell Sorter. Introduction

Application Note 10. Measurement of Cell Recovery. After Sorting with a Catcher-Tube-Based. Cell Sorter. Introduction Application Note 10 Measurement of Cell Recovery After Sorting with a Catcher-Tube-Based Cell Sorter Introduction In many experiments using sorted cells, it is important to be able to count the number

More information

Institut für Experimentelle Immunologie und Bildgebung

Institut für Experimentelle Immunologie und Bildgebung Page 1 von 6 Starting Up the System Start up the computer, start Windows and log in as IMCES_user start BD FACSDiva software with your Groupname Turn on the cytometer main power Wait a moment and Check

More information

TABLE OF CONTENT. Page ACKNOWLEDGEMENTS. iii ENGLISH ABSTRACT THAI ABSTRACT. vii LIST OF TABLES LIST OF FIGURES. xvi ABBREVIATIONS.

TABLE OF CONTENT. Page ACKNOWLEDGEMENTS. iii ENGLISH ABSTRACT THAI ABSTRACT. vii LIST OF TABLES LIST OF FIGURES. xvi ABBREVIATIONS. x TABLE OF CONTENT ACKNOWLEDGEMENTS ENGLISH ABSTRACT THAI ABSTRACT LIST OF TABLES LIST OF FIGURES ABBREVIATIONS iii iv vii xv xvi xviii CHAPTER I: INTRODUCTION 1.1 Statement of problems 1 1.2 Literature

More information

JC-1 Mitochondrial Membrane Potential

JC-1 Mitochondrial Membrane Potential Introduction Background Apoptosis is a cellular process involving a genetically programmed series of events leading to the death of a cell. During this process, several key events occur in mitochondria,

More information

PROTOCOL. Immunocytochemistry (ICC) MATERIALS AND EQUIPMENT REQUIRED

PROTOCOL. Immunocytochemistry (ICC) MATERIALS AND EQUIPMENT REQUIRED PROTOCOL Immunocytochemistry (ICC) 1850 Millrace Drive, Suite 3A Eugene, Oregon 97403 11-07 MATERIALS AND EQUIPMENT REQUIRED Materials: MitoSciences primary monoclonal antibody/antibodies Fluorophore-conjugated

More information

Lab 02: Blood Cytology (20 points)

Lab 02: Blood Cytology (20 points) Pierce College Putman/Biol 242 Name: Lab 02: Blood Cytology (20 points) Reference: Marieb & Mitchell 9 th Ed: 29A (Activities 1, 2, 3, 4, 7); 10 th Ed: Exercise 29 (Activities 1, 2, 3, 4, 7). Pierce College

More information

123count ebeads Catalog Number: 01-1234 Also known as: Absolute cell count beads GPR: General Purpose Reagents. For Laboratory Use.

123count ebeads Catalog Number: 01-1234 Also known as: Absolute cell count beads GPR: General Purpose Reagents. For Laboratory Use. Page 1 of 1 Catalog Number: 01-1234 Also known as: Absolute cell count beads GPR: General Purpose Reagents. For Laboratory Use. Normal human peripheral blood was stained with Anti- Human CD45 PE (cat.

More information

Classic Immunoprecipitation

Classic Immunoprecipitation 292PR 01 G-Biosciences 1-800-628-7730 1-314-991-6034 technical@gbiosciences.com A Geno Technology, Inc. (USA) brand name Classic Immunoprecipitation Utilizes Protein A/G Agarose for Antibody Binding (Cat.

More information

INSTRUCTION Probemaker

INSTRUCTION Probemaker INSTRUCTION Probemaker Instructions for Duolink In Situ Probemaker PLUS (Art. no. 92009-0020) and Duolink In Situ Probemaker MINUS (Art. no. 92010-0020) Table of content 1. Introduction 4 2. Applications

More information

Outline. 1. Experiment. 2. Sample analysis and storage. 3. Image analysis and presenting data. 4. Probemaker

Outline. 1. Experiment. 2. Sample analysis and storage. 3. Image analysis and presenting data. 4. Probemaker Tips and tricks Note: this is just an informative document with general recommendations. Please contact support@olink.com should you have any queries. Document last reviewed 2011-11-17 Outline 1. Experiment

More information

Introduction to Flow Cytometry:

Introduction to Flow Cytometry: Introduction to Flow Cytometry: A Learning Guide Manual Part Number: 11-11032-01 April, 2000 BD Biosciences 2350 Qume Drive San Jose, CA 95131-1807 1-800-448-2347 Introduction to Flow Cytometry: A Learning

More information

General western blot protocol. Guidance for running an efficient and accurate experiment

General western blot protocol. Guidance for running an efficient and accurate experiment blot protocol Guidance for running an efficient and accurate experiment Contents Introduction Solution and reagents Sample lysis Sample preparation Loading and running the gel Antibody staining Useful

More information

NCL Method ITA-14. Analysis of Nanoparticle Effects on Maturation of Monocyte Derived Dendritic Cells In Vitro

NCL Method ITA-14. Analysis of Nanoparticle Effects on Maturation of Monocyte Derived Dendritic Cells In Vitro NCL Method ITA-14 Analysis of Nanoparticle Effects on Maturation of Monocyte Derived Dendritic Cells In Vitro Nanotechnology Characterization Laboratory Frederick National Laboratory for Cancer Research

More information

Amaxa Mouse T Cell Nucleofector Kit

Amaxa Mouse T Cell Nucleofector Kit Amaxa Mouse T Cell Nucleofector Kit For T cells isolated from C57BL/6 & BALB/c mice Evaluated for murine T cells isolated from C57BL/6 & BALB/c mice This protocol is designed for murine lymphocytes or

More information

Flow cytometry basics fluidics, optics, electronics...

Flow cytometry basics fluidics, optics, electronics... Title Flow cytometry basics fluidics, optics, electronics... RNDr. Jan Svoboda, Ph.D. Cytometry and Microscopy Core Facility IMB, CAS, v.v.i Vídeňská 1083 Fluorescence Fluorescence occurs when a valence

More information

Application Information Bulletin: Set-Up of the CytoFLEX Set-Up of the CytoFLEX* for Extracellular Vesicle Measurement

Application Information Bulletin: Set-Up of the CytoFLEX Set-Up of the CytoFLEX* for Extracellular Vesicle Measurement Application Information Bulletin: Set-Up of the CytoFLEX Set-Up of the CytoFLEX* for Extracellular Vesicle Measurement Andreas Spittler, MD, Associate Professor for Pathophysiology, Medical University

More information

International Beryllium Conference, Montreal, Canada March 10, 2005

International Beryllium Conference, Montreal, Canada March 10, 2005 Alternative Lymphocyte Proliferation Tests: BrdU and Flow Cytometry Based Tests International Beryllium Conference, Montreal, Canada March 10, 2005 Tim K. Takaro Department of Environmental and Occupational

More information

Cell Cycle Tutorial. Contents

Cell Cycle Tutorial. Contents Cell Cycle Tutorial Contents Experimental Requirements...2 DNA Dyes...2 Protocols...3 PI Parameter & Analysis Setup...4 PI Voltage Adjustments...6 7-AAD Parameter Setup...6 To-Pro3 Parameter Setup...6

More information

Sysmex UF-1000i and UF-500i: the modern age of urinalysis

Sysmex UF-1000i and UF-500i: the modern age of urinalysis Sysmex UF-1000i and UF-500i: the modern age of urinalysis Sysmex Xtra Online February 2011 The request for a urine status in the laboratory routine mostly concerns incoming orders within the scope of health

More information

Serology: Fluorescent antibody tests and other tests employing conjugated antibodies

Serology: Fluorescent antibody tests and other tests employing conjugated antibodies Serology: Fluorescent antibody tests and other tests employing conjugated antibodies Authors: Adapted by Prof M van Vuuren. Originally compiled by Dr RW Worthington. (Retired) Licensed under a Creative

More information

Results: A plot showing propidium iodide and Annexin is used to determine apoptotic (Annexin only +) from necrotic (PI+ and Annexin +).

Results: A plot showing propidium iodide and Annexin is used to determine apoptotic (Annexin only +) from necrotic (PI+ and Annexin +). Results: A plot showing propidium iodide and Annexin is used to determine apoptotic (Annexin only +) from necrotic (PI+ and Annexin +). This group should be a low percentage. Surface markers can then be

More information

HighPure Maxi Plasmid Kit

HighPure Maxi Plasmid Kit HighPure Maxi Plasmid Kit For purification of high pure plasmid DNA with high yields www.tiangen.com PP120109 HighPure Maxi Plasmid Kit Kit Contents Storage Cat.no. DP116 Contents RNaseA (100 mg/ml) Buffer

More information

Hoechst 33342 HSC Staining and Stem Cell Purification Protocol (see Goodell, M., et al. (1996) J Exp Med 183, 1797-806)

Hoechst 33342 HSC Staining and Stem Cell Purification Protocol (see Goodell, M., et al. (1996) J Exp Med 183, 1797-806) Hoechst 33342 HSC Staining and Stem Cell Purification Protocol (see Goodell, M., et al. (1996) J Exp Med 183, 1797-86) The Hoechst purification was established for murine hematopoietic stem cells (HSC)

More information

FLOW CYTOMETRY: PRINCIPLES AND APPLICATIONS. By: Douaa Moh. Sayed

FLOW CYTOMETRY: PRINCIPLES AND APPLICATIONS. By: Douaa Moh. Sayed FLOW CYTOMETRY: PRINCIPLES AND APPLICATIONS By: Douaa Moh. Sayed Definition Flow cytometry is a technique for counting, examining, and sorting microscopic particles suspended in a stream of fluid. It allows

More information

TECHNICAL BULLETIN. FluoroTag FITC Conjugation Kit. Product Number FITC1 Storage Temperature 2 8 C

TECHNICAL BULLETIN. FluoroTag FITC Conjugation Kit. Product Number FITC1 Storage Temperature 2 8 C FluoroTag FITC Conjugation Kit Product Number FITC1 Storage Temperature 2 8 C TECHNICAL BULLETIN Product Description The FluoroTag FITC Conjugation Kit is suitable for the conjugation of polyclonal and

More information

LAB 14 ENZYME LINKED IMMUNOSORBENT ASSAY (ELISA)

LAB 14 ENZYME LINKED IMMUNOSORBENT ASSAY (ELISA) STUDENT GUIDE LAB 14 ENZYME LINKED IMMUNOSORBENT ASSAY (ELISA) GOAL The goal of this laboratory lesson is to explain the concepts and technique of enzyme linked immunosorbent assay (ELISA). OBJECTIVES

More information

THE UNIVERSITY OF NEWCASTLE- DISCIPLINE OF MEDICAL BIOCHEMISTRY. STANDARD OPERATING PROCEDURE PROCEDURE NO: GLP 079 MOD: 1st Issue Page: 1 of 7

THE UNIVERSITY OF NEWCASTLE- DISCIPLINE OF MEDICAL BIOCHEMISTRY. STANDARD OPERATING PROCEDURE PROCEDURE NO: GLP 079 MOD: 1st Issue Page: 1 of 7 THE UNIVERSITY OF NEWCASTLE- DISCIPLINE OF MEDICAL BIOCHEMISTRY STANDARD OPERATING PROCEDURE PROCEDURE NO: GLP 079 MOD: 1st Issue Page: 1 of 7 Procedure Type: Title: General Laboratory Procedure Cell Counts

More information

Chapter 6: Antigen-Antibody Interactions

Chapter 6: Antigen-Antibody Interactions Chapter 6: Antigen-Antibody Interactions I. Strength of Ag-Ab interactions A. Antibody Affinity - strength of total noncovalent interactions between single Ag-binding site on an Ab and a single epitope

More information

Ficoll-Paque PREMIUM Ficoll-Paque PREMIUM 1.084 Ficoll-Paque PREMIUM 1.073

Ficoll-Paque PREMIUM Ficoll-Paque PREMIUM 1.084 Ficoll-Paque PREMIUM 1.073 Instructions 28-4039-56 AE Cell Preparation Media Ficoll-Paque PREMIUM Ficoll-Paque PREMIUM 1.084 Ficoll-Paque PREMIUM 1.073 Intended use For in vitro isolation of mononuclear cells and/or granulocytes

More information

Photosynthesis Biology 100 - Concepts of Biology 5.1

Photosynthesis Biology 100 - Concepts of Biology 5.1 Photosynthesis Biology 100 - Concepts of Biology 5.1 Name Instructor Lab Section Objectives: To gain an understanding of: Leaf structure The nature of light and pigments (eg, chlorophyll) How the wavelength

More information

BD FACSComp Software Tutorial

BD FACSComp Software Tutorial BD FACSComp Software Tutorial This tutorial guides you through a BD FACSComp software lyse/no-wash assay setup run. If you are already familiar with previous versions of BD FACSComp software on Mac OS

More information

Zenon Labeling Technology A New Approach to Immunolabeling from Molecular Probes

Zenon Labeling Technology A New Approach to Immunolabeling from Molecular Probes Zenon Labeling Technology A New Approach to Immunolabeling from Molecular Probes Applications for Flow Cytometry and Imaging A New Approach to Immunolabeling Zenon labeling technology provides a versatile,

More information

COMPENSATION MIT Flow Cytometry Core Facility

COMPENSATION MIT Flow Cytometry Core Facility COMPENSATION MIT Flow Cytometry Core Facility Why do we need compensation? 1) Because the long emission spectrum tail of dyes causes overlap like with the fluorophores FITC and PE. 2) For sensitivity reasons,

More information

Affinity Chromatography

Affinity Chromatography PR100-02 G-Biosciences 1-800-628-7730 1-314-991-6034 technical@gbiosciences.com A Geno Technology, Inc. (USA) brand name Affinity Chromatography Teacher s Guidebook (Cat. # BE-417) think proteins! think

More information

Human Peripheral Blood Mononuclear Cell (PBMC) Manual

Human Peripheral Blood Mononuclear Cell (PBMC) Manual Human Peripheral Blood Mononuclear Cell (PBMC) Manual INSTRUCTION MANUAL ZBM0063.04 SHIPPING CONDITIONS Human Peripheral Blood Mononuclear Cells, cryopreserved Cryopreserved human peripheral blood mononuclear

More information

Standardization, Calibration and Quality Control

Standardization, Calibration and Quality Control Standardization, Calibration and Quality Control Ian Storie Flow cytometry has become an essential tool in the research and clinical diagnostic laboratory. The range of available flow-based diagnostic

More information

PRODUCT INFORMATION SHEET Monoclonal antibodies detecting human antigens

PRODUCT INFORMATION SHEET Monoclonal antibodies detecting human antigens www.iqproducts.nl PRODUCT INFORMATION SHEET Monoclonal antibodies detecting human antigens IFN- γ PURE [RUO] [REF] IQP-160P s 50 tests FITC [RUO] [REF] IQP-160F s 50 tests R-PE [RUO] [REF] IQP-160R s 50

More information

Using BD FACSDiva CST To. Evaluate Cytometer Performance, Create Custom Assay Settings. and

Using BD FACSDiva CST To. Evaluate Cytometer Performance, Create Custom Assay Settings. and Using BD FACSDiva CST To Evaluate Cytometer Performance, Create Custom Assay Settings and Implement Cross-Instrument and Cross-Site Standardization of Assays PART 2 Alan M. Stall Director, Advanced Cytometry

More information

Measuring Cell Viability/Cytotoxicity: Cell Counting Kit-F

Measuring Cell Viability/Cytotoxicity: Cell Counting Kit-F Introduction The Cell Counting Kit-F is a fluorometic assay for the determination of viable cell numbers. Calcein-AM in this kit passes through the cell membrane and is hydrolized by the esterase in the

More information

Multicolor Flow Cytometry: Setup and Optimization on the BD Accuri C6 Flow Cytometer

Multicolor Flow Cytometry: Setup and Optimization on the BD Accuri C6 Flow Cytometer Multicolor Flow Cytometry: Setup and Optimization on the BD Accuri C6 Flow Cytometer Presented by Clare Rogers, MS Senior Marketing Applications Specialist BD Biosciences 23-13660-00 Webinar Overview Multicolor

More information

Too Many B Cells: Chronic Lymphocytic Leukemia and the Role of Flow Cytometry

Too Many B Cells: Chronic Lymphocytic Leukemia and the Role of Flow Cytometry Too Many B Cells: Chronic Lymphocytic Leukemia and the Role of Flow Cytometry by Debby R. Walser-Kuntz Biology Department Carleton College, Northfield, MN Taylor goes in to see her doctor, Dr. Chavez,

More information

LIVE/DEAD Fixable Dead Cell Stain Kits

LIVE/DEAD Fixable Dead Cell Stain Kits USER GUIDE LIVE/DEAD Fixable Dead Cell Stain Kits Pub. No. MAN0002416 (MP34955) Rev. A.0 Table 1. Contents and storage Material Amount Storage Stability Individual Kits: Blue, violet, aqua, yellow-, green,

More information

Protocol v002 Page 1 of 7 Agencourt RNAdvance Cell v2 Total RNA Isolation from Cultured Cells

Protocol v002 Page 1 of 7 Agencourt RNAdvance Cell v2 Total RNA Isolation from Cultured Cells Page 1 of 7 Agencourt RNAdvance Cell v2 Total RNA Isolation from Cultured Cells Please refer to http://www.agencourt.com/technical for updated protocols and refer to MSDS instructions http://www.beckmancoulter.com/customersupport/msds/msds.asp

More information

Protein transfer from SDS-PAGE to nitrocellulose membrane using the Trans-Blot SD cell (Western).

Protein transfer from SDS-PAGE to nitrocellulose membrane using the Trans-Blot SD cell (Western). Western Blot SOP Protein transfer from SDS-PAGE to nitrocellulose membrane using the Trans-Blot SD cell (Western). Date: 8/16/05, 10/31/05, 2/6/06 Author: N.Oganesyan, R. Kim Edited by: R. Kim Summary:

More information

HARVESTING AND CRYOPRESERVATION OF HUMAN EMBRYONIC STEM CELLS (hescs)

HARVESTING AND CRYOPRESERVATION OF HUMAN EMBRYONIC STEM CELLS (hescs) HARVESTING AND CRYOPRESERVATION OF HUMAN EMBRYONIC STEM CELLS (hescs) OBJECTIVE: can be cryopreserved in a liquid nitrogen (LN 2 ) freezer for long-term storage. This Standard Operating Procedure (SOP)

More information

General Annexin V Staining Procedure

General Annexin V Staining Procedure UCANU 0007 Version 01, November 2011 Page 1 of 6 CMCI (Center for molecular and Cellular Intervention) University Medical Center Utrecht Written By Name Function Date Signature Mark Klein Lab manager Conformation

More information

IMMUNOFLUORESCENCE MODULE 62.1 INTRODUCTION. Notes

IMMUNOFLUORESCENCE MODULE 62.1 INTRODUCTION. Notes Immunofluorescence MODULE 62 IMMUNOFLUORESCENCE 62.1 INTRODUCTION Immunofluorescence (IF) is one of the very common laboratory techniques used in almost all disciplines of Biology including Medicine for

More information

For in vitro preparation of human mononuclear cells from peripheral blood, bone marrow, and umbilical cord blood. Not for in vitro diagnostic use.

For in vitro preparation of human mononuclear cells from peripheral blood, bone marrow, and umbilical cord blood. Not for in vitro diagnostic use. GE Healthcare Instructions 28-4039-56 AA Cell Preparation Media Ficoll-Paque PREMIUM Intended use For in vitro preparation of human mononuclear cells from peripheral blood, bone marrow, and umbilical cord

More information

Instructions. Torpedo sirna. Material. Important Guidelines. Specifications. Quality Control

Instructions. Torpedo sirna. Material. Important Guidelines. Specifications. Quality Control is a is a state of the art transfection reagent, specifically designed for the transfer of sirna and mirna into a variety of eukaryotic cell types. is a state of the art transfection reagent, specifically

More information

Titering Antibodies INTRODUCTION Materials Procedure for titering antibodies to extracellular antigens A. Directly conjugated antibodies

Titering Antibodies INTRODUCTION Materials Procedure for titering antibodies to extracellular antigens A. Directly conjugated antibodies C.C. Stewart, S.J. Stewart, Titering Antibodies. In: Current Protocols in Cytometry, (J.P. Robinson, Z. Darzynkiewicz, P. Dean. L. Dressler, P.Rabinovitch, C. Stewart, H. Tanke, L. Wheeless, eds.) J.Wiley

More information

Workshop 14-16 February 2006

Workshop 14-16 February 2006 Theoretical and practical approaches of Hepatocyte primary culture Workshop 14-16 February 2006 Lecture (2) Disaggregation & purification of target cells Coarse organizer Dr. Abo bakr Mohamed Eltayeb General

More information

Mouse IFN-gamma ELISpot Kit

Mouse IFN-gamma ELISpot Kit Page 1 of 8 Mouse IFN-gamma ELISpot Kit Without Plates With Plates With Sterile Plates Quantity Catalog Nos. 862.031.001 862.031.001P 862.031.001S 1 x 96 tests 862.031.005 862.031.005P 862.031.005S 5 x

More information

Uses of Flow Cytometry

Uses of Flow Cytometry Uses of Flow Cytometry 1. Multicolour analysis... 2 2. Cell Cycle and Proliferation... 3 a. Analysis of Cellular DNA Content... 4 b. Cell Proliferation Assays... 5 3. Immunology... 6 4. Apoptosis... 7

More information

Annexin V-FITC Apoptosis Detection Kit

Annexin V-FITC Apoptosis Detection Kit ab14085 Annexin V-FITC Apoptosis Detection Kit Instructions for Use For the rapid, sensitive and accurate measurement of Apoptosis in living cells (adherent and suspension). This product is for research

More information

Using the BD TM Cytometer Setup and Tracking (CS&T) System for Instrument Characterization and Performance Tracking

Using the BD TM Cytometer Setup and Tracking (CS&T) System for Instrument Characterization and Performance Tracking Using the BD TM Cytometer Setup and Tracking (CS&T) System for Instrument Characterization and Performance Tracking Mark KuKuruga Senior Technical Applications Specialist BD Biosciences 23-14462-00 Outline

More information

FITC Annexin V/Dead Cell Apoptosis Kit with FITC annexin V and PI, for Flow Cytometry

FITC Annexin V/Dead Cell Apoptosis Kit with FITC annexin V and PI, for Flow Cytometry FITC Annexin V/Dead Cell Apoptosis Kit with FITC annexin V and PI, for Flow Cytometry Catalog no. V13242 Table 1. Contents and storage information. Material Amount Composition Storage* Stability FITC annexin

More information

Purification by Recrystallization

Purification by Recrystallization Experiment 2 Purification by Recrystallization Objectives 1) To be able to select an appropriate recrystallizing solvent. 2) To separate and purify acetanilide by recrystallization. 3) To compare the melting

More information

Protein extraction from Tissues and Cultured Cells using Bioruptor Standard & Plus

Protein extraction from Tissues and Cultured Cells using Bioruptor Standard & Plus Protein extraction from Tissues and Cultured Cells using Bioruptor Standard & Plus Introduction Protein extraction from tissues and cultured cells is the first step for many biochemical and analytical

More information

Mouse krebs von den lungen 6 (KL-6) ELISA

Mouse krebs von den lungen 6 (KL-6) ELISA KAMIYA BIOMEDICAL COMPANY Mouse krebs von den lungen 6 (KL-6) ELISA For the quantitative determination of mouse KL-6 in serum, plasma, cell culture supernatants, body fluid and tissue homogenate Cat. No.

More information

DAY 0 IN VITRO FERTILIZATION

DAY 0 IN VITRO FERTILIZATION DAY 0 IN VITRO FERTILIZATION INITIAL PREPARATION FOR SPERM PURIFICATION AND FERTILIZATION Laminar flow hood ISOLATE SOF-FERT H-SOF 15 ml conical centrifuge tubes 3 centrifuge carriers Dish, 35mm x 10 mm

More information