Method to deplete carryover white blood cells from CELLSEARCH Epithelial Cell Profile Kit samples
|
|
- Marian Thompson
- 7 years ago
- Views:
Transcription
1 Technical Notes Method to deplete carryover white blood cells from CELLSEARCH Epithelial Cell Profile Kit samples Introduction This paper details a method to deplete carryover white blood cells from samples following CTC enrichment when using the CELLSEARCH Epithelial Cell Profile Kit. Circulating tumor cells (CTCs) of epithelial origin are present in the blood of cancer patients at very low frequency (< 10 / ml blood). In order to characterize CTCs, they need to be enriched from large volumes of blood (7.5 mls). The CELLSEARCH Circulating Tumor Cell (Epithelial) assay uses magnetic particles conjugated to antiepithelial cell adhesion molecule (EpCAM) to selectively capture CTCs. After enrichment, the sample contains CTCs and a number ( ) of white blood cells (WBCs). The presence of WBCs in the CTC enriched sample may interfere with the molecular analysis of CTC biomarkers, especially if it is coexpressed by WBCs. We have improved the ability to perform molecular characterization of CTCs by selectively depleting WBCs carried over during CTC enrichment. The CELLSEARCH Epithelial Cell Profile Kit enriches CTCs without permeabilization or staining of cells. Investigators must confirm the utility of these methods for their intended application. Principle of Operation Samples after processing on the CELLTRACKS AUTOPREP SYSTEM using the CELLSEARCH Epithelial Cell Profile Kit are labeled with antibody to the leukocyte marker CD45. The anti-cd45 antibody used is conjugated to fluorescein isothiocyanate (FITC). The anti-cd45-fitc binds to the WBCs carried over into the CTC enriched cell population. Samples are incubated for 1 hour in microtiter plates coated with anti-fitc antibody. The anti-fitc binds the FITC present on the anti-cd45-fitc and causes the CD45 positive WBCs to adhere to the plate. The CD45 negative CTCs remain unbound, and are subsequently harvested with the supernatant fluids. Figure 1 shows the principle of white blood cell separation from tumor cells after tumor cells enrichment in the CELLSEARCH Circulating Epithelial Cell Kit. Page 1 of 8
2 Figure 1: Principle of WBC depletion EpCAM FF EpCAM FF WBC CD45-FITC Tumor Cell FITC WBC Tumor Cell Anti FITC Materials Required 1. Anti FITC from Novus (catalog # NB ) 2. CD45-FITC (Becton-Dickinson or Biolegend 3. R&D Systems Cellect Magnet #MAG997 (Magnetic separator) 4. 6 well plate from VWR (Falcon #353046) 5. PBS/1%BSA (BSA from Seracare # AP or equivalent BSA from other vendor) 6. 12x75mm 5ml polystyrene round bottom tube BD# Method A. Coating of 6 well plate with anti FITC 1. Prepare 50mM bicarbonate buffer ph 8.5. Prepare buffer fresh same day before use. 2. Dilute anti FITC from Novus in 50mM bicarbonate buffer (step 1) to 50ug/ml. Coating each well requires 800ul of anti FITC solution. Prepare sufficient anti FITC solution to coat at least one well per sample to be WBC depleted. Page 2 of 8
3 3. Add 800ul of anti FITC to each well and make sure the entire surface of the well is completely covered with no bubbles or empty spots. 4. Incubate at Room Temperature (RT) for 3 hours, followed by overnight incubation at 2-8 o C (Cover the plate with the lid during all the incubations). 5. After overnight incubation, warm the plate to RT for 30minutes and aspirate all the supernatant. 6. Rinse each well with PBS twice using 1ml for each rinse and aspirate the supernatant after the 2 nd rinse. 7. Block each well with 1ml of PBS/1%BSA to prevent any non-specific binding of CD45-FITC or cells to the plate. 8. Incubate the plate at RT for 4 hours. 9. Aspirate all the supernatant and rinse the plate twice with 2ml/rinse of PBS. 10. Aspirate all the buffer and air dry the plate at RT for one hour. 11. Cover the plate with the lid and store the plate at 2-8 o C until use. 12. Plate may be stored at 2-8 o C for up to 30 days before use. Other storage conditions or times must be validated by the investigator. B. Labeling of cells with CD45-FITC 1. Prepare the samples using CELLSEARCH Epithelial Cell Profile kits 2. Take the sample out of the CELLTRACKS AUTOPREP SYSTEM and transfer the entire sample (~ 900ul) to a 12x75mm polystyrene tube. 3. Add CD45-FITC to the sample to final concentration of 2ug/ml and mix by gently vortexing. 4. Incubate the sample in the dark for 15 minutes at RT. 5. Remove excess unbound CD45-FITC by washing as follows. 6. Add 3ml of PBS/1%BSA to the sample, vortex gently and place the sample in the magnetic separator. 7. After 15 minutes, and with the sample tube still in the magnetic separator, aspirate the supernatant using a glass Pasteur pipette or plastic transfer pipettes. Discard the supernatant fluid. 8. Remove the tube from the magnet and wash the sample again by repeating steps 6 and 7 above. 9. Remove the tube from the magnet, add 900ul of PBS/1% BSA and vortex gently. Page 3 of 8
4 C. Application of sample to 6 well plate coated with anti FITC 1. Bring the anti FITC coated plate to RT (minimum of 30 minutes) on the day of use. 2. Add 2ml of PBS/1% BSA to the wells and incubate for 15minutes. 3. Aspirate the buffer and rinse the well once more with PBS/1%BSA 4. Aspirate all of the buffer before sample is added to the well. 5. Add the entire sample from the step B-9 to one of the wells of 6 well plate. 6. Incubate the sample for 1 hour in the dark at RT. Every 15 minutes, gently rock the plate forward and back 3x (no more than a 45 degree angle) to mix the sample. 7. After 1 hour incubation, gently rock the plate forward and back 3x (no more than a 45 degree angle) 8. Keep the plate tilted at a 45 degree angle, aspirate the sample using 1ml pipette and dispense it at the top of the well to get all the unbound cells into the sample. Repeat this aspiration and dispension step 2 more times and then transfer the entire sample (~ 900ul) from each well to a desired tube for downstream molecular analysis. Preliminary results: The method described above was tested with normal EDTA blood spiked with tissue cultured tumor cells. 7.5ml of EDTA blood spiked with SKBR3 cells was processed on the CELLTRACKS AUTOPREP SYSTEM using a CELLSEARCH Epithelial Cell Profile Kit. An enriched sample (900ul) from the CELLTRACKS AUTOPREP SYSTEM using a CELLSEARCH sample tube was transferred to a 12x75mm tube. After labeling the cells with CD45-FITC, the excess CD45-FITC was removed by washing the sample 2-times with 3ml of PBS/1%BSA by magnetic separation for 15minutes. The final sample was resuspended in 900ul of PBS/1%BSA and applied to the wells coated with anti-fitc. The samples were incubated for 1 hour with gentle mixing every 15 minutes. After one hour, the supernatant was removed from the wells and the number of white blood cells and SKBR3 cells were determined by flow cytometry. The cells were labeled with anti-her2neu conjugated to allophycocyanin dye (APC) to detect SKBR3 cells by flow cytometry. The white blood cells were detected in the FITC channel since they were labeled with anti-cd45-fitc. The number of white blood cells and SKBR3 cells present in the samples was calculated before and after depletion step. The cell count present before depletion was normalized to 100% to calculate percent cell depletion.the results from this study are shown in the Figure 2. Page 4 of 8
5 Figure 2: Percent Removal of white blood and tumor cells by anti-fitc coated plate Cell Depletion (%) WBC Dep (%) SKBR3 Dep (%) Ave. Number of Samples Tested Flow cytometry results show that greater than 85% of the CTCs were recovered in the supernatant fluids, while greater than 90% of WBCs had been depleted. These results were then confirmed by molecular analysis. Molecular measurements of WBC depletion in CELLSEARCH Epithelial Cell Profile kit enriched samples Duplicate 7.5ml of EDTA blood from 6 healthy donors was processed on the CELLTRACKS AUTOPREP SYSTEM using a CELLSEARCH Epithelial Cell Profile Kit. Following CELLTRACKS AUTOPREP SYSTEM enrichment, one tube from each donor was subjected to either the Depleted method which removed the WBC from the sample or the Non-depleted method which serves as a control to measure the WBC contamination in the sample without the depletion process. Non-Depleted method Samples are removed from the CELLTRACKS AUTOPREP SYSTEM and placed in a magnet for 15 minutes. The buffer is aspirated and the cells are lysed in RLT buffer (Qiagen). Depleted method Following CELLTRACKS AUTOPREP SYSTEM enrichment, the excess CD45-FITC was removed by washing the sample 2-times with 3ml of PBS/1%BSA by magnetic separation for 15minutes. The final sample was resuspended in 900ul of PBS/1%BSA and then applied to the wells coated with anti-fitc. Page 5 of 8
6 The samples were incubated for 1 hour with gentle mixing every 15minutes. After one hour, the supernatant was removed from the wells and the cells were placed in a magnet for 15 minutes. The buffer was removed and the cells were lysed in RLT buffer (Qiagen). For both the depleted and non depleted samples, the RNA was purified using the Qiagen AllPrep Kit and reverse transcribed using the High Capacity cdna Reverse Transcription Kits kit (Life Technologies). Complimentary DNA was amplified using TaqMan PreAmp Master Mix Kit (Life Technologies) and primer sets as for genes listed in the figures below. Quantitative PCR was carried out on the amplified samples for two genes known to be expressed in white blood cells (CD45 (aka PTPRC) and BST1) as well as a housekeeping gene expressed (B-Actin). Efficacy of WBC depletion was measured by the loss of WBC and housekeeping specific gene signals in the depleted sample relative to the non-depleted sample. Figure 3 - Molecular measurement of WBC removal. Circles and squares represent individual measurements for the non-depleted and depleted samples respectively. Lines represent the mean measurement for each sample type. P values represent the results of student s T-tests performed between the depleted and non-depleted samples for each gene measured. Page 6 of 8
7 T-tests between the depleted and non-deleted samples showed a significant loss of WBC specific and housekeeping genes as a result of CD45 depletion. Mean delta Ct measurements of >2 cycles indicate a >75% reduction of the WBC signals in the CD45 depleted samples. Improvement of CTC detection following white blood cell depletion One potential advantage of the CD45 depletion method is an improved ability to detect CTC or characterize CTC gene expression when there is some level of background gene expression contributed by the white blood cells. Reducing the number of white blood cells should reduce the background gene expression and improve the ability to detect transcripts in lower numbers of CTC. To demonstrate this principle four samples were prepared for each of six donors; two samples were spiked with 10 VCaP cells and two samples were unspiked. One spiked and unspiked sample was prepared with both the Depleted and Non-depleted protocol as described above. RNA extraction, Reverse Transcription and Pre-Amplification kit were carried out as above. Public databases were mined to identify a panel of potential CTC markers that could be useful for measuring utility of the WBC depletion method. Genes were selected that had moderate to high expression in VCaP cells and lower expression in WBC. p = 0.09 p <0.001 p = 0.24 p =0.01 Figure 4 Improved detection of CTC markers following CD45 depletion. RT-PCR results for potential CTC markers S100A13 and AKR1C3. Without WBC depletion (non-depleted samples) both genes show no significant difference in expression between unspiked and 10-cell spiked sample sets. In the depleted set the background contributed by WBC is reduced and there is a significant difference between the unspiked and 10-cell spike sample sets. Page 7 of 8
8 Conclusions: The white blood cell depletion method demonstrates that the cell population following WBC removal is significantly depleted of WBC with minimal loss of cancer cells. Thus, this method has a potential to improve the detection of tumor-specific markers in clinical samples enriched for the presence of circulating tumor cells. The decision on whether to implement the WBC depletion protocol will depend on the background expression level of the specific genes of interest. The depletion method may not be necessary for genes that have no background expression in WBC. For genes that have high levels of expression in WBC, it is likely that signals from WBC will mask signals from CTC despite WBC depletion method. It is likely that the method will be most useful in improving the detectability of markers that have a low level of background expression in WBC. It is recommended that the depletion method be tested on healthy donors for each gene of interest to determine the WBC background level and benefit of the procedure. References: 1. Chandra Rao, Brad Foulk, Karl Nielsen, Bhavesh Vaidya, Jaymala Patel, Dana Gaffney, Denis Smirnov, Jayaprakash Karkera and Mark Connelly Janssen Research & Development, LLC, Huntingdon Valley, PA Improved Molecular Characterization of Circulating Tumor Cells AACR 2013 Abstract #1443 Page 8 of 8
How To Get A Cell Print
QUICK CELL CAPTURE AND CHARACTERIZATION GUIDE FOR CELLSEARCH CUSTOMERS CellSave EDTA Blood sample Rare cell capture Enumeration Single protein marker Cell capture for molecular characterization CELLSEARCH
More informationRNA Extraction and Quantification, Reverse Transcription, and Real-time PCR (q-pcr)
RNA Extraction and Quantification, Reverse Transcription, and Real-time Preparation of Samples Cells: o Remove media and wash cells 2X with cold PBS. (2 ml for 6 well plate or 3 ml for 6cm plate) Keep
More informationHow To Get Rid Of Small Dna Fragments
AxyPrep TM Mag FragmentSelect-I Protocol (Fragment Size Selection for Illumina Genome Analyzer and Life Technologies SoLiD) Introduction The AxyPrep Mag FragmentSelect-I purification kit utilizes a unique
More informationAxyPrep TM Mag PCR Clean-up Protocol
AxyPrep TM Mag PCR Clean-up Protocol Intro The AxyPrep Mag PCR Clean-up kit utilizes a unique paramagnetic bead technology for rapid, high-throughput purification of PCR amplicons. Using this kit, PCR
More informationNimbleGen DNA Methylation Microarrays and Services
NimbleGen DNA Methylation Microarrays and Services Sample Preparation Instructions Outline This protocol describes the process for preparing samples for NimbleGen DNA Methylation microarrays using the
More informationRPCI 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 informationStandard 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 informationReal-time quantitative RT -PCR (Taqman)
Real-time quantitative RT -PCR (Taqman) Author: SC, Patti Lab, 3/03 This is performed as a 2-step reaction: 1. cdna synthesis from DNase 1-treated total RNA 2. PCR 1. cdna synthesis (Advantage RT-for-PCR
More informationMLX BCG Buccal Cell Genomic DNA Extraction Kit. Performance Characteristics
MLX BCG Buccal Cell Genomic DNA Extraction Kit Performance Characteristics Monolythix, Inc. 4720 Calle Carga Camarillo, CA 93012 Tel: (805) 484-8478 monolythix.com Page 2 of 9 MLX BCG Buccal Cell Genomic
More informationPCR and Sequencing Reaction Clean-Up Kit (Magnetic Bead System) 50 preps Product #60200
3430 Schmon Parkway Thorold, ON, Canada L2V 4Y6 Phone: 866-667-4362 (905) 227-8848 Fax: (905) 227-1061 Email: techsupport@norgenbiotek.com PCR and Sequencing Reaction Clean-Up Kit (Magnetic Bead System)
More informationab139418 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 informationEdU 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 informationApplication Guide... 2
Protocol for GenomePlex Whole Genome Amplification from Formalin-Fixed Parrafin-Embedded (FFPE) tissue Application Guide... 2 I. Description... 2 II. Product Components... 2 III. Materials to be Supplied
More informationMinimal 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 informationab185916 Hi-Fi cdna Synthesis Kit
ab185916 Hi-Fi cdna Synthesis Kit Instructions for Use For cdna synthesis from various RNA samples This product is for research use only and is not intended for diagnostic use. Version 1 Last Updated 1
More informationUser Manual. CelluLyser Lysis and cdna Synthesis Kit. Version 1.4 Oct 2012 From cells to cdna in one tube
User Manual CelluLyser Lysis and cdna Synthesis Kit Version 1.4 Oct 2012 From cells to cdna in one tube CelluLyser Lysis and cdna Synthesis Kit Table of contents Introduction 4 Contents 5 Storage 5 Additionally
More informationChromatin Immunoprecipitation
Chromatin Immunoprecipitation A) Prepare a yeast culture (see the Galactose Induction Protocol for details). 1) Start a small culture (e.g. 2 ml) in YEPD or selective media from a single colony. 2) Spin
More informationTaqman TCID50 for AAV Vector Infectious Titer Determination
Page 1 of 8 Purpose: To determine the concentration of infectious particles in an AAV vector sample. This process involves serial dilution of the vector in a TCID50 format and endpoint determination through
More informationAmaxa 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 informationProtocol 001298v001 Page 1 of 1 AGENCOURT RNACLEAN XP IN VITRO PRODUCED RNA AND CDNA PURIFICATION
Page 1 of 1 AGENCOURT RNACLEAN XP IN VITRO PRODUCED RNA AND CDNA PURIFICATION Please refer to http://www.agencourt.com/technical for updated protocols and refer to MSDS instructions when handling or shipping
More informationCellCelector : enabling fast automated image-based isolation of pure single CTCs for personalized therapy
short version CellCelector : enabling fast automated image-based isolation of pure single CTCs for personalized therapy Titre Dr. Constantin Nelep - September, 2014 1 Who we are ALS Headquarters in Jena,
More informationIgM ELISA. For the quantitative determination of IgM in human serum and plasma. For Research Use Only. Not For Use In Diagnostic Procedures.
IgM ELISA For the quantitative determination of IgM in human serum and plasma For Research Use Only. Not For Use In Diagnostic Procedures. Please read carefully due to Critical Changes, e.g., Calibrator
More informationRealLine HCV PCR Qualitative - Uni-Format
Instructions for use PCR KIT FOR EXTRACTION OF RNA AND REAL TIME PCR DETECTION KIT FOR HEPATITIS C VIRUS RNA Research Use Only Qualitative Uni Format VBD0798 48 tests valid from: December 2013 Rev11122013
More informationGenomic DNA Extraction Kit INSTRUCTION MANUAL
Genomic DNA Extraction Kit INSTRUCTION MANUAL Table of Contents Introduction 3 Kit Components 3 Storage Conditions 4 Recommended Equipment and Reagents 4 Introduction to the Protocol 4 General Overview
More informationChromatin Immunoprecipitation (ChIP)
Chromatin Immunoprecipitation (ChIP) Day 1 A) DNA shearing 1. Samples Dissect tissue (One Mouse OBs) of interest and transfer to an eppendorf containing 0.5 ml of dissecting media (on ice) or PBS but without
More informationProcedure for RNA isolation from human muscle or fat
Procedure for RNA isolation from human muscle or fat Reagents, all Rnase free: 20% SDS DEPC-H2O Rnase ZAP 75% EtOH Trizol Chloroform Isopropanol 0.8M NaCitrate/1.2M NaCl TE buffer, ph 7.0 1. Homogenizer-probe
More informationLAB 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 informationInstructions. 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 informationDNA Isolation Kit for Cells and Tissues
DNA Isolation Kit for Cells and Tissues for 10 isolations of 00 mg each for tissue or 5 x 10 7 cultured cells Cat. No. 11 81 770 001 Principle Starting material Application Time required Results Benefits
More informationDynabeads mrna DIRECT Micro Kit
USER GUIDE Dynabeads mrna DIRECT Micro Kit Catalog Number 61021 Revision 004 Revision Date 14 May 2012 For Research Use Only. Not for human or animal therapeutic or diagnostic use. For Research Use Only.
More informationHiPer RT-PCR Teaching Kit
HiPer RT-PCR Teaching Kit Product Code: HTBM024 Number of experiments that can be performed: 5 Duration of Experiment: Protocol: 4 hours Agarose Gel Electrophoresis: 45 minutes Storage Instructions: The
More informationQUANTITATIVE RT-PCR. A = B (1+e) n. A=amplified products, B=input templates, n=cycle number, and e=amplification efficiency.
QUANTITATIVE RT-PCR Application: Quantitative RT-PCR is used to quantify mrna in both relative and absolute terms. It can be applied for the quantification of mrna expressed from endogenous genes, and
More informationHBV Quantitative Real Time PCR Kit
Revision No.: ZJ0002 Issue Date: Aug 7 th, 2008 HBV Quantitative Real Time PCR Kit Cat. No.: HD-0002-01 For Use with LightCycler 1.0/LightCycler2.0/LightCycler480 (Roche) Real Time PCR Systems (Pls ignore
More informationClassic 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 informationncounter Gene Expression Assay Manual Total RNA and Cell Lysate Protocols
ncounter Gene Expression Assay Manual Total RNA and Cell Lysate Protocols v.20090807 For research use only. Not for use in diagnostic procedures. Limited License Subject to the terms and conditions of
More informationHuman Free Testosterone(F-TESTO) ELISA Kit
Human Free Testosterone(F-TESTO) ELISA Kit Catalog Number. MBS700040 For the quantitative determination of human free testosterone(f-testo) concentrations in serum, plasma. This package insert must be
More informationTransformAid Bacterial Transformation Kit
Home Contacts Order Catalog Support Search Alphabetical Index Numerical Index Restriction Endonucleases Modifying Enzymes PCR Kits Markers Nucleic Acids Nucleotides & Oligonucleotides Media Transfection
More informationWestern Blot Protocol Protein isolation
Western Blot Protocol Protein isolation A. Preparation of cell lysates. - Preparation of materials: -Dial the microcentrifuge temperature control setting to 4 C -Prepare a bucket of ice -Prepare lysis
More informationMaxwell 16 Blood DNA Purification System
Technical Manual Maxwell 16 Blood DNA Purification System Caution: Handle cartridges with care; seal edges may be sharp. 2800 Woods Hollow Rd. Madison, WI USA In Vitro Diagnostic Medical Device MDSS GmbH
More informationMystiCq microrna cdna Synthesis Mix Catalog Number MIRRT Storage Temperature 20 C
microrna cdna Synthesis Mix Catalog Number MIRRT Storage Temperature 20 C Product Description The microrna cdna Synthesis Mix has been designed to easily convert micrornas into cdna templates for qpcr
More informationIntroduction To Real Time Quantitative PCR (qpcr)
Introduction To Real Time Quantitative PCR (qpcr) SABiosciences, A QIAGEN Company www.sabiosciences.com The Seminar Topics The advantages of qpcr versus conventional PCR Work flow & applications Factors
More informationPROTOCOL. 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 informationWHOLE 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 informationArcturus PicoPure RNA Isolation Kit. User Guide
Arcturus PicoPure RNA Isolation Kit User Guide For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. Information in this document is subject to change without notice.
More informationRecommended Procedures for the Extraction of RNA. Jan Pedersen USDA, APHIS, VS, National Veterinary Services Laboratories, Ames, IA 50010
Recommended Procedures for the Extraction of RNA Jan Pedersen USDA, APHIS, VS, National Veterinary Services Laboratories, Ames, IA 50010 RNA Extraction Isolates RNA from other cellular components in the
More informationTaqMan Fast Advanced Master Mix. Protocol
TaqMan Fast Advanced Master Mix Protocol For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. Information in this document is subject to change without notice. APPLIED
More informationHow To Use An Enzymatics Spark Dna Sample Prep Kit For Ion Torrent
SPARK DNA Sample Prep Kit Ion Torrent (SPK0002-V08) Frequently Asked Questions Under what circumstances would I use SPARK DNA Sample Prep Kit for Ion Torrent? Enzymatics SPARK DNA Sample Prep Kit for Ion
More informationGenolution Pharmaceuticals, Inc. Life Science and Molecular Diagnostic Products
Genolution Pharmaceuticals, Inc. Revolution through genes, And Solution through genes. Life Science and Molecular Diagnostic Products www.genolution1.com TEL; 02-3010-8670, 8672 Geno-Serum Hepatitis B
More information50 g 650 L. *Average yields will vary depending upon a number of factors including type of phage, growth conditions used and developmental stage.
3430 Schmon Parkway Thorold, ON, Canada L2V 4Y6 Phone: 866-667-4362 (905) 227-8848 Fax: (905) 227-1061 Email: techsupport@norgenbiotek.com Phage DNA Isolation Kit Product # 46800, 46850 Product Insert
More informationqstar mirna qpcr Detection System
qstar mirna qpcr Detection System Table of Contents Table of Contents...1 Package Contents and Storage Conditions...2 For mirna cdna synthesis kit...2 For qstar mirna primer pairs...2 For qstar mirna qpcr
More informationLab 2. Isolation of mononuclear cells from peripheral blood and separation into subpopulations
Lab 2 Isolation of mononuclear cells from peripheral blood and separation into subpopulations Supervisors: Sissela Broos sissela.broos@immun.lth.se tel: 222 96 78 Niclas Olsson niclas.olsson@immun.lth.se
More informationBovine Vitamin B12 (VB12) ELISA Kit
Bovine Vitamin B12 (VB12) ELISA Kit Catalog Number. CSB-E14089B For the quantitative determination of endogenic bovine vitamin B12 (VB12) concentrations in serum, plasma, tissue homogenates. This package
More informationFastLine cell cdna Kit
1. FastLine cell cdna Kit For high-speed preparation of first-strand cdna directly from cultured cells without RNA purification www.tiangen.com RT100701 FastLine cell cdna Kit Cat. no. KR105 Kit Contents
More informationImmunoglobulin E (IgE) concentrations in Human. Immunoglobulin E (IgE) Human ELISA Kit
ab108650 Immunoglobulin E (IgE) Human ELISA Kit Instructions for Use For the quantitative measurement of Immunoglobulin E (IgE) concentrations in Human serum. This product is for research use only and
More informationMouse IgM ELISA. Cat. No. KT-407 K-ASSAY. For the quantitative determination of IgM in mouse biological samples. For Research Use Only. 1 Rev.
K-ASSAY Mouse IgM ELISA For the quantitative determination of IgM in mouse biological samples Cat. No. KT-407 For Research Use Only. 1 Rev. 072309 K-ASSAY PRODUCT INFORMATION Mouse IgM ELISA Cat. No. KT-407
More informationCovalent Conjugation to Cytodiagnostics Carboxylated Gold Nanoparticles Tech Note #105
Covalent Conjugation to Cytodiagnostics Carboxylated Gold Nanoparticles Tech Note #105 Background Gold nanoparticle conjugates have been widely used in biological research and biosensing applications.
More informationImmunophenotyping 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 informationOptimized Protocol sirna Test Kit for Cell Lines and Adherent Primary Cells
page 1 of 8 sirna Test Kit for Cell Lines and Adherent Primary Cells Kit principle Co-transfection of pmaxgfp, encoding the green fluorescent protein (GFP) from Pontellina p. with an sirna directed against
More informationQuickTiter Hepatitis B Surface Antigen (HBsAg) ELISA Kit
Product Manual QuickTiter Hepatitis B Surface Antigen (HBsAg) ELISA Kit Catalog Numbers VPK-5004 VPK-5004-5 96 assays 5 x 96 assays FOR RESEARCH USE ONLY Not for use in diagnostic procedures Introduction
More informationCytoSelect 48-Well Cell Adhesion Assay (ECM Array, Fluorometric Format)
Product Manual CytoSelect 48-Well Cell Adhesion Assay (ECM Array, Fluorometric Format) Catalog Number CBA-071 CBA-071-5 48 assays 5 x 48 assays FOR RESEARCH USE ONLY Not for use in diagnostic procedures
More informationpcas-guide System Validation in Genome Editing
pcas-guide System Validation in Genome Editing Tagging HSP60 with HA tag genome editing The latest tool in genome editing CRISPR/Cas9 allows for specific genome disruption and replacement in a flexible
More informationMagExtractor -Genome-
Instruction manual MagExtractor-Genome-0810 F0981K MagExtractor -Genome- NPK-101 100 preparations Store at 4 C Contents [1] Introduction [2] Components [3] Materials required [4] Protocol 1. Purification
More informationCancer Antigen CA125 Human ELISA Kit
ab108653 Cancer Antigen CA125 Human ELISA Kit Instructions for Use For the quantitative measurement of Human Cancer Antigen CA125 concentrations in serum. This product is for research use only and is not
More informationApplication Note. Single Cell PCR Preparation
Application Note Single Cell PCR Preparation From Automated Screening to the Molecular Analysis of Single Cells The AmpliGrid system is a highly sensitive tool for the analysis of single cells. In combination
More informationRat Creatine Kinase MB isoenzyme,ck-mb ELISA Kit
Rat Creatine Kinase MB isoenzyme,ck-mb ELISA Kit Catalog No: E0479r 96 Tests Operating instructions www.eiaab.com FOR RESEARCH USE ONLY; NOT FOR THERAPEUTIC OR DIAGNOSTIC APPLICATIONS! PLEASE READ THROUGH
More informationUltraClean Forensic DNA Isolation Kit (Single Prep Format)
UltraClean Forensic DNA Isolation Kit (Single Prep Format) Catalog No. Quantity 14000-10 10 preps 14000-S 1 prep Instruction Manual Please recycle Version: 10302012 1 Table of Contents Introduction...
More informationCompleteⅡ 1st strand cdna Synthesis Kit
Instruction Manual CompleteⅡ 1st strand cdna Synthesis Kit Catalog # GM30401, GM30402 Green Mountain Biosystems. LLC Web: www.greenmountainbio.com Tel: 800-942-1160 Sales: Sales@ greenmountainbio.com Support:
More informationThe fastest spin-column based procedure for purifying up to 10 mg of ultra-pure endotoxin-free transfection-grade plasmid DNA.
INSTRUCTION MANUAL ZymoPURE Plasmid Gigaprep Kit Catalog Nos. D4204 (Patent Pending) Highlights The fastest spin-column based procedure for purifying up to 10 mg of ultra-pure endotoxin-free transfection-grade
More informationFirst Strand cdna Synthesis
380PR 01 G-Biosciences 1-800-628-7730 1-314-991-6034 technical@gbiosciences.com A Geno Technology, Inc. (USA) brand name First Strand cdna Synthesis (Cat. # 786 812) think proteins! think G-Biosciences
More informationAgencourt RNAdvance Blood Kit for Free Circulating DNA and mirna/rna Isolation from 200-300μL of Plasma and Serum
SUPPLEMENTAL PROTOCOL WHITE PAPER Agencourt RNAdvance Blood Kit for Free Circulating DNA and mirna/rna Isolation from 200-300μL of Plasma and Serum Bee Na Lee, Ph.D., Beckman Coulter Life Sciences Process
More informationOutline. 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 informationSOLIDscript Solid Phase cdna Synthesis Kit Instruction Manual
Toll Free: 866-252-7771 752A Lincoln Blvd. Phone: 732-469-7771 Fax: 732-469-7782 Middlesex, NJ 08846 Web: www.purebiotechllc.com SOLIDscript Solid Phase cdna Synthesis Kit Instruction Manual Product: SOLIDscript
More informationGRS Plasmid Purification Kit Transfection Grade GK73.0002 (2 MaxiPreps)
1 GRS Plasmid Purification Kit Transfection Grade GK73.0002 (2 MaxiPreps) (FOR RESEARCH ONLY) Sample : Expected Yield : Endotoxin: Format : Operation Time : Elution Volume : 50-400 ml of cultured bacterial
More informationSerology: 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 informationHiPer Ion Exchange Chromatography Teaching Kit
HiPer Ion Exchange Chromatography Teaching Kit Product Code: HTC001 Number of experiments that can be performed: 5 Duration of Experiment: Protocol: 5-6 hours Storage Instructions: The kit is stable for
More informationPROTOCOL. 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 informationData Sheet. PD-L1:B7-1[Biotinylated] Inhibitor Screening Assay Kit Catalog # 72026 Size: 96 reactions
Data Sheet PD-L1:B7-1[Biotinylated] Inhibitor Screening Assay Kit Catalog # 72026 Size: 96 reactions BACKGROUND: There have been a number of studies defining a role for PD-L1 in the regulation of immune
More informationFITC 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 informationBelow 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 informationHuman IP-10 ELISA Kit, pink-one
1. Catalog No. K0331210P 2. Quantity 96 tests 3. Storage 4 C Human IP-10 ELISA Kit, pink-one 4. Description Human IP-10 ELISA kit contains all the necessary reagents required for performing quantitative
More informationUltraClean Soil DNA Isolation Kit
PAGE 1 UltraClean Soil DNA Isolation Kit Catalog # 12800-50 50 preps New improved PCR inhibitor removal solution (IRS) included Instruction Manual (New Alternative Protocol maximizes yields) Introduction
More informationHuman Luteinizing Hormone (LH) Custom Kit
Human Luteinizing Hormone (LH) Custom Kit 18089-v2-2015Jan 1 MSD Toxicology Assays Human Luteinizing Hormone (LH) Custom Kit This package insert must be read in its entirety before using this product.
More informationHARVESTING 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 informationMouse Creatine Kinase MB isoenzyme (CKMB) ELISA
KAMIYA BIOMEDICAL COMPANY Mouse Creatine Kinase MB isoenzyme (CKMB) ELISA For the quantitative determination of mouse CKMB in serum, plasma, cell culture fluid and other biological fluids Cat. No. KT-57681
More informationIntroduction 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 informationempcr Amplification Method Manual - Lib-A
GS Junior Titanium Series May 2010 (Rev. April 2011) For life science research only. Not for use in diagnostic procedures. 1. WORKFLOW The emulsion-based clonal amplification (empcr amplification) of a
More informationHow Does a Doctor Test for AIDS?
Edvo-Kit #S-70 How Does a Doctor Test for AIDS? S-70 Experiment Objective: The Human Immunodefi ciency Virus (HIV) is an infectious agent that causes Acquired Immunodefi ciency Syndrome (AIDS) in humans.
More informationSDS-PAGE Protocol Mutated from the SDS-PAGE protocol written by the Lord of the Flies
SDS-PAGE Protocol Mutated from the SDS-PAGE protocol written by the Lord of the Flies Pouring the resolving gel 1. Clean glass plates with soap and water, then with ethanol. Assemble the glass plates and
More informationABSTRACT. Promega Corporation, Updated September 2008. http://www.promega.com/pubhub. 1 Campbell-Staton, S.
A Modified Wizard SV Genomic DNA Purification System Protocol to Purify Genomic DNA... A Modified Wizard SV Genomic DNA Purification System Protocol to Purify Genomic DNA from Shed Reptile Skin ABSTRACT
More informationProtein 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 informationPlant Genomic DNA Extraction using CTAB
Plant Genomic DNA Extraction using CTAB Introduction The search for a more efficient means of extracting DNA of both higher quality and yield has lead to the development of a variety of protocols, however
More informationKevin Bogart and Justen Andrews. Extraction of Total RNA from Drosophila. CGB Technical Report 2006-10 doi:10.2506/cgbtr-200610
Kevin Bogart and Justen Andrews Extraction of Total RNA from Drosophila CGB Technical Report 2006-10 doi:10.2506/cgbtr-200610 Bogart K and Andrews J. 2006. Extraction of Total RNA from Drosophila. CGB
More informationResults: 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 informationNo-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 informationBlood Collection and Processing SOP
Brisbane Breast Bank Blood Collection and Processing SOP Breast Pathology Laboratory University of Queensland Centre for Clinical Research Blood Collection We collect 30ml of blood from patients who have
More information竞 争 性 分 析 Epitope Mapping 实 验 方 法
竞 争 性 分 析 Epitope Mapping 实 验 方 法 ABSTRACT The simplest way to determine whether two monoclonal antibodies bind to distinct sites on a protein antigen is to carry out a competition assay. The assay can
More informationMir-X mirna First-Strand Synthesis Kit User Manual
User Manual Mir-X mirna First-Strand Synthesis Kit User Manual United States/Canada 800.662.2566 Asia Pacific +1.650.919.7300 Europe +33.(0)1.3904.6880 Japan +81.(0)77.543.6116 Clontech Laboratories, Inc.
More informationMouse 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 informationBD PuraMatrix Peptide Hydrogel
BD PuraMatrix Peptide Hydrogel Catalog No. 354250 Guidelines for Use FOR RESEARCH USE ONLY NOT FOR CLINICAL, DIAGNOSTIC OR THERAPEUTIC USE SPC-354250-G rev 2.0 1 TABLE OF CONTENTS Intended Use... 2 Materials
More informationTechnical Manual No. 0173 Update Date 10112010
TissueDirect TM Multiplex PCR System Technical Manual No. 0173 Update Date 10112010 I Description.. 1 II Applications 2 III Key Features.. 2 IV Shipping and Storage. 3 V Simplified Procedures. 3 VI Detailed
More information