BD FACSCalibur Flow Cytometer Cleaning Procedure

Transcription

1 CTCR Core Facility 1 University of Delaware BD FACSCalibur Flow Cytometer Cleaning Procedure Please run the complete cleaning procedure every time you finish the acquisition of your samples. 1. To wash the FACS Analyzers a) Load a tube containing ~3ml 10% bleach onto the SIP (sample injection port) with support arm to the side and leave for one minute on Run and Hi. b) Using the same tube, place the arm under the tube and hit Run and Hi for 5 minutes. c) Remove the bleach tube and replace with a tube containing DD water. d) Repeat step a) using DD water. e) Repeat step b) for 5 minutes with DD water (wash II). f) Leave the tube with 1 ml of DD Water (wash II) on STANDBY. (The DD Water should remain in the tube when the instrument is on STANDBY or has been turned off to prevent salt deposits from forming in the sample injection tube.) 2. Refill the sheath reservoir up to no more than 3/4 full (see detailed instructions on the next page). The FACSFlow SHEATH solution is ready to use (Becton Dickinson, Cat. # ) and is located by the sink. Additional FACSFlow SHEATH supply is stored next to the door to your left. (Ensure that fluid hoses and red wire are not twisted when reinstalling the reservoir). 3. Empty the waste reservoir (see detailed instructions below). 4. Remove all items remaining from your experiment. Wipe the bench top, an ethanol spray bottle is available. Quit the CellQuest software, LOG OFF the multi-user system and shut down the computer and FACS. Collect any printed pages from the printer and push in the chairs. Double check to be sure you have collected all of your items before leaving. Filling the SHEATH RESERVOIR: 1. If the FACS Analyzer (FACSCalibur) is powered on, put it on STANDBY. 2. Slide out the fluidics drawer. If the FACS Analyzer is powered on, please do not forget to relieve the pressure by pushing the vent valve toggle switch. 3. Slide the black metal bracket over the sheath reservoir away from you, and lift up to remove it. 4. Unscrew the cap assembly from the sheath reservoir and set the assembly aside on the clean paper towel, placed on the top of waste reservoir. 5. Remove the sheath reservoir. 6. Fill the sheath reservoir to mark on the outside of the container with the FACSFlow SHEATH solution located by the sink. (CAUTION: when the sheath reservoir is filled beyond the recommended level, fluid may backflow and damage the instrument as well as preventing the system from pressurizing correctly.) 7. Install the sheath reservoir into the fluidics drawer. (CAUTION: before you install the reservoir back into the fluidics drawer, wipe the outside of the sheath reservoir with clean paper towel to avoid the corrosion of the instrument.) 8. Replace and tighten the cap assembly. (CAUTION: try to avoid twists and pinches of the tubing.) 9. Replace the black metal bracket and re-pressurize the system by flipping the toggle switch. Always empty the waste reservoir when you fill the sheath reservoir. It will prevent the waste reservoir from overflowing.

2 CTCR Core Facility 2 University of Delaware Emptying the WASTE RESERVOIR: 1. Unscrew the cap assembly from the waste reservoir and set the assembly aside on the clean paper towel, placed on the top of sheath reservoir. 2. Remove the waste reservoir. 3. Empty the waste reservoir into the sink. Let the tap water run for 1-2 minutes. Rinse the waste reservoir with tap water. Except if the samples were stained with Propdium iodide (PI), in this case the waste should be transferred into an appropriate waste container located on the floor on the left side of the FACS station. 4. Put approximately 200ml of straight bleach into the waste tank. Commercial (96 oz) container with bleach is stored under the sink. 5. Install the waste reservoir back into the fluidics drawer. (CAUTION: before you install the reservoir back into the fluidics drawer, wipe the outside of the waste reservoir with clean paper towel to avoid the corrosion of the instrument.) 6. Replace and tighten the cap assembly. (CAUTION: try to avoid twists and pinches of the tubing.) 7. If the FACS Analyzer is powered on, please do not forget to pressurize the sheath reservoir by pushing the vent valve toggle switch (follow the arrow sign). 8. Check the sheath line for bubbles, if bubbles are visible, push the roller in the roller clamp forward to allow the pressurized sheath fluid to force the air bubbles into the waste tank and return the roller clamp to the closed position. Repeat if necessary. 9. Check the sheath filter line for bubbles, if any are visible then disconnect the sheath filter tubing at the quick-disconnect port and press the tip of the valve against the side of a waste container to force the bubbles out with the flow of the fluid. Gently tap the filter to see if any other bubbles are inside, repeat if necessary. Reconnect the line gently by holding the quick disconnect open while reattaching the line to avoid introducing new bubbles. 10. Slide the fluidics drawer back into the FACS system. Useful Tips to Improve Sample Quality for the Flow Cytometric Analysis and Cell Sorting (Live Cells) Low cellular viability, auto-fluorescence, and cell aggregates will result in poor flow cytometric analysis and sorts. Improved cell preparation will result in better sort purity, yield, and cell viability 1) Make all cell preparations strictly on ice, unless otherwise stated in the procedure 2) Buffer suggestions a) Use Ca/Mg++ free buffers (for example: DPBS, 1X without calcium & magnesium, Sterile, Cat# CV, Mediatech, Inc) b) Use BSA (0.1-1%) or dialyzed FBS (1-5%) (any source will be good) for the protein support of the buffer. Addition of non-dialyzed serum to the buffers will replace the Ca/Mg++. Use lowest sufficient concentration of BSA to improve data quality (decrease auto-fluorescence and increase population resolution) c) Use EDTA at up to 5mM concentration (for example: ready to use 0.02% in DPBS (0.5 mm), Cat# E8008, Sigma-Aldrich Co.; or ~0.5 M in H 2 O, Cat#03690, Sigma-Aldrich Co.). It may help to prevent cation-dependent cell-cell adhesion

3 CTCR Core Facility 3 University of Delaware d) Add 25-50ug/ml of DNAse I (Cat# D4513, Sigma-Aldrich Co.) and 5mM MgCl 2 (Magnesium chloride hexahydrate, Cat# M2670, Sigma-Aldrich Co.) to the buffer. It may help if cells are clumping due to the cell death 3) Single cell suspension a) At any stage syringe the sample through the 25 gauge needle, 3-5 slow passages b) Filter samples just prior to analysis or cell sorting to remove remaining clumps. The 12 x 75-mm tube with the cell-strainer cap ("blue-cap tubes"; #161900, RU Stockroom; or Cat#352235, BD Falcon) offers the convenient way to filter laboratory samples. Note: It's important to prevent clumping (using tips above) before you filter your samples through a nylon mesh, otherwise you will lose too many cells 4) Keep cells at reasonable concentration of million/ml, dependent on the cell type. Avoid keeping cells at unnecessary high concentration for the prolonged periods of time 5) Use dead cell exclusion/discrimination dyes to "eliminate" dead cells from analysis and sort. Choice of dead cell exclusion dye depends on the color combination of fluorochromes within the sample a) For non-fixed cells use conventional dead cell exclusion dyes, for example - PI, 7AAD, DAPI, SYTOX Blue, Green, or Red; etc. Note: DO NOT USE conventional dead cell exclusion dyes (listed above) on the fixed cells b) For fixed cells - use fixable dead cell exclusion dyes, for example - LIVE/DEAD Fixable Blue, Aqua, Violet, Green, or Red; etc. Avoid: 1. Vigorous vortexing. It could damage fragile cells 2. Excessive and unnecessary centrifuge spin. Use minimal necessary forces (rpm) to sediment cells 3. Aspirating the entire buffer, leaving dry pellet of cells. Cells will die in the dry pellet 4. Bubbles. In bubbles due to the surface tension forces cells tend to die Sample Guidelines for the Cell Sort 1) Human samples must be serology tested before brought to the FCRC 2) Samples suppose to be single cell suspension (SCS) only (i.e. no clumps). 3) If you have extensive cell death in the sample, add DNAse. It will not only decrease clump formation in the sample, but also will decrease viscosity of the single cell suspension buffer, by digestion of the free DNA. Sort purity and yield could go substantially up after DNAase treatment 4) Add the 10-25mM of HEPES, ph range (for example: HEPES buffer solution 1M in H 2 O, Sigma-Aldrich Co., Cat# ML-F) to your buffer. Addition of HEPES will significantly increase the buffer capacity of the original sample buffer. Buffer capacity of the common phosphate and carbonate buffers gets compromised by high pressure within the instrument during the cell sorting procedure 5) Provide compensation controls: a) If cells are used as single color controls i) Unstained cells ii) All separate single color stained cell controls (1) Preferable concentration of the controls is around 1 x 10 6 cells/ml, volume ml

4 CTCR Core Facility 4 University of Delaware b) If beads are used as single color controls i) Unstained Cells (1) Preferable concentration of unstained cell control is around 1 x 10 6 cells/ml, volume ml ii) Unstained beads iii) All separate single color stained bead controls 6) Provide gating controls - Fluorescent Minus One (FMO) controls, as proper way to evaluate the background in certain channel and set sorting gates appropriately 7) Sample concentration for the sort should typically be around x 10 6 cells/ml. If you have fewer then 5 x 10 6 cells put them into the minimal volume of 500 ul 8) Provide extra 15 ml of the buffer you used for the cell samples 9) Bulk Sort - Provide collection tubes for the sorted cells. Tube size depends on the expected number of post-sorted cells. Note: Concentration of post-sorted cells is between x 10 6 cells/ml and is depends on the nozzle size a) Tube types: i) Screw Cap Micro Tube (up to 2.0 ml) (1) 1.5 ml - sterile (Cat# , VWR) (2) 1.5 ml - low retention tubes (Cat# , VWR) (3) 2.0 ml - sterile (Cat# , VWR) ii) 12 x 75 mm tubes (up to 4.5 ml) (1) Polypropylene - best choice (a) sterile with snap cap (Cat# A, Fisher Scientific; or Cat#352063, BD Biosciences) (2) Polystyrene - poorer choice, but still possible (a) non-sterile (Cat#352008, BD Biosciences) (b) sterile with the cap (Cat#352054, BD Biosciences) iii) 15 ml tubes - any type b) Tube pre-treatment: i) In order to prevent cells sticking to the sides of the tubes, pre-coat the tubes, filling them with 1% BSA ii) Keep filled tubes inverted for at least 30' prior sort c) Solution to sort into: i) Trysol (for RNA Sorts) ii) PBS-based buffer iii) PBS/HEPES/BSA (see buffer suggestions) iv) Any specific solution (for example, PCR mix) v) none d) Tube special characteristics depend on the sorting purpose: i) For sterile sort tubes should be also sterile ii) For RNA work tubes should be RNAse free iii) For Western blot tubes should not be treated with external protein 10) Sort using ACDU (automated cell deposition unit): a) Receptacles: i) Multiwell plates: (1) 96 well plate (2) 24 well plate ii) PCR Tubes in stripes iii) Microscope slides

5 CTCR Core Facility 5 University of Delaware iv) Etc. b) Solution to sort into - depends on the purpose of the sort: i) Complete medium for cell growth ii) Trysol iii) PCR mix iv) Etc. c) Sort setup specifics: i) Day before the sort FCRC Staff should be provided with several empty plates (or other receptacles) of the type planned to be used for the sort ii) Plates (or other receptacles) for the cell sort should be brought to FCRC at the time specified by FCRC Staff (when sort is completely pre-set using empty receptacles) Bead Compensation Controls Reason to use BD FACSComp Bead as Single Stain Controls: a) Cell numbers are limiting b) Antigen is dimly expressed c) Antibody has low affinity to receptor d) Positive population is rare e) Use tandem dyes BD FACSComp Bead Kits. Details and ordering information: 1) Each Kit contains two vials with bead polystyrene particles; 6ml each; $ a. Beads uncoated (FBS) that have no binding capacity; used for negative population b. Beads coated with Goat anti-ig kappa which bind light chain-bearing immunoglobulin; used for positive population. Be sure to use same Fluorochromeconjugated monoclonal antibodies as actually used for cell staining in your experiment 2) Three BD FACSComp Bead Kits available with the coated beads of different specificity: a. Anti-Mouse Ig, kappa (Cat#552843, BD Biosciences) b. Anti-Rat Ig, kappa (Cat#552844, BD Biosciences) c. Anti-Rat/Hamster Ig, kappa (Cat#552845, BD Biosciences) BD FACSComp Beads labeling procedure (implemented and recommended by FCRC): 1) Verify origin of your antibody (Hamster, Rat or Mouse) to specify which beads should be used 2) Mix Beads well by shaking the bottle 3) Pre- dilute anti- Ig Beads using 400 ul of PBS for 1 drop of Bead 4) Dispense 100 ul of pre- diluted Beads per single color control sample a. Add 5 ul of pre- diluted primary Ab to the Beads 5) Incubate 20 min at RT, mix occasionally 6) If applicable, add 5 ul of pre- diluted secondary Ab with Fluorochrome 7) Incubate 20 min at RT, mix occasionally 8) Add 50 um of pre- diluted uncoated Beads to each sample 9) Make separate tube with uncoated beads for Negative Control 10) Use immediately for Multi- color compensation or store at +4 o C for up to 2 weeks