Flow cytometry

Scope

1. This standard operating procedure describes a multiparametric flow cytometry assay used to measure specific cell surface and intracellular expression of markers of T cell activation and exhaustion on conventional and regulatory human T-lymphocytes.  This procedure is written for use in staining peripheral blood mononuclear cells (PBMC). 

1. DEFINITIONS AND Abbreviations
   1. Ab                   antibody
   2. APC                allophycocyanin
   3. BV                  Brilliant Violet
   4. ddH₂O            deionized, distilled water
   5. DMSO            dimethyl sulfoxide
   6. FACS              fluorescence activated cell sorting
   7. FBS                 fetal bovine serum
   8. FITC               fluorescein isothiocyanate
   9. PBMC             peripheral blood mononuclear cells
   10. PBS                 phosphate-buffered saline
   11. PE                   phycoerythrin
   12. PerCP              peridinin chlorophyll protein
   13. QC                  quality control
   14. RP10               RPMI cell culture media containing 10% FBS
   15. RT                   room temperature (20-25°C)
   16. TC                   tissue culture

1. Materials and reagents

1. Equipment

1. PROCEDURE
   1. Buffer Preparation 
      1. FACS Buffer (prepare prior to experiment)
         1. Prepare FACS Buffer for cell washes and staining (1% FBS and 0.05% sodium azide) by adding 10 ml FBS and 5 ml 10% sodium azide to 1 L PBS.  This is good stored at 4°C for up to 3 months.  Dispense cold FACS buffer into a 50-mL polypropylene tube and keep on ice.
      2. 1X Permeabilization Buffer (prepare prior to experiment)
         1. Dilute 5 mL of 10X Permeabilization buffer with 45 mL dH₂0.  The 1X final working solution contains 0.1% saponin and 0.009% sodium azide.  The 1X Permeabilization Buffer should be used to resuspend and wash the cell pellet (after cells have been fixed).  Store at 4°C. 

1. Thawing and Counting of Cryopreserved PBMC Samples
   1. Thaw cryopreserved PBMC samples to be tested.  Each stain will require 0.5 – 1 x 10⁶ cells, and there are 3 stains required per sample: 2 stains with the specific antibody cocktail and 1 stain with the isotype control antibody cocktail (thus, ~1.5 – 3x10⁶ cells/sample total). 
   2. After thawing, count cells and record viability.
   3. Using the final, dilution-adjusted cell concentration for each sample, resuspend original tube of cells at 5x10⁶/ml in RP10 and proceed with antibody staining.

1. Surface Antibody Cocktail Preparation

1. NOTE: Keep antibodies protected from light and in cold storage (2-8°C) before and after use.  Prepare cocktail after cell thawing.  Surface and intracellular antibody volumes and dilutions listed were optimized for the specific lot used during assay development and may change for new lots of antibodies.
2. Prepare a master staining cocktail using the pre-determined appropriate dilutions of each antibody. For each sample, 50 µl of antibody cocktail will be added directly to pelleted PBMC. Calculate volume of each antibody needed and subtract total volume by total antibody volume to determine how much FACS Buffer to add to bring up to final volume.  Be sure to prepare ~20% extra for volume losses.  Keep antibody cocktail covered on ice until ready for use. 
3. See example below for details for a 12-sample staining experiment (12 duplicate stains with specific Ab cocktail + 12 single stains with isotype control cocktail).  Scale as necessary based on number of samples and replicate stains, and prepare extra for volume losses.
4. NOTE: For Qdot antibodies, centrifuge for 1-2 min at 4000 rpm (894 rcf) in microcentrifuge or quick spin mini-centrifuge prior to use to pellet any particulate materials. Be sure to take aliquot only from top portion of Qdot antibody vial to avoid particulates. All other antibodies should be briefly vortexed prior to use. When making the antibody master mix, make this in reagent reservoirs and avoid using plastic microcentrifuge tubes that may absorb the Qdot antibody.  Antibodies should be added to FACS buffer already distributed into the reservoir to prevent possible adherence of concentrated antibodies to the reservoir. 

1. Surface Staining of Cryopreserved PBMC Samples
   1. Add 0.5-1x10⁶ cells (100-200 ml of cells resuspended at 5x10⁶ cells/ml) from each PBMC sample to one well per stain in an appropriately labeled 96-well V-bottom plate using a predetermined plate layout for where samples are to be mapped on the plate (see assay worksheet).  Each sample will require 3 separate wells of 5x10⁵ cells each for duplicate stains with specific panel antibodies and 1 single stain with isotype control antibodies.  If needed, add PBS to each well to bring to a 200 µl final volume in each well. 
   2. Centrifuge plate at 900xg (~2000 rpm) in tabletop centrifuge with plate adapter for 3 min to pellet cells at 4°C.
   3. Decant supernatant by flicking 96-well plate upside down into the sink. Keeping the plate inverted, blot gently on a paper towel to ensure minimal retention of supernatant. 
   4. Wash cells by adding 200 µl/well of PBS. Centrifuge plate at 900xg (~2000 rpm) at 4°C in a tabletop centrifuge for 3 min to pellet cells.
   5. Prepare a master mix of sufficient volume of Live/Dead-Aqua fixable dead cell stain in PBS at 0.2 µl per 50 µl volume stain (1:250 final dilution) to stain all wells and add 50 µl per well.  Mix well and incubate cells for 15 minutes at room temperature protected from light. 
   6. Wash cells by adding 150 µl/well of FACS buffer. Centrifuge plate at 900xg (~2000 rpm) at 4°C in a tabletop centrifuge for 3 min to pellet cells.
   7. Decant supernatant by flicking 96-well plate upside down into the sink. Keeping the plate inverted, blot gently on a paper towel to ensure minimal retention of supernatant.
   8. Add 50 µl/well of prepared surface staining antibody cocktail for each duplicate stain per sample and add 50 µl/well of the surface isotype control antibody cocktail to a single replicate of each sample (total of 3 stains per sample in separate wells). Ensure thorough mixing by pipetting up and down several times.
   9. Cover plate in the dark and leave at 4°C in the refrigerator for exactly 30 min for incubation with antibodies.
2. Fixation and Permeabilization of Stained PBMC Samples
   1. Prepare Fixation/Permeabilization Buffer:
      1. Dilute 1 part Fixation/Permeabilization Concentrate (4X stock solution must be used within 6 months of opening) with 3 parts Fixation/Permeabilization Diluent.  For example: 40 samples @ 150 µl/sample = 6000 µl total volume needed. You would dilute 1500 µl Concentrate with 4500 µl diluent to obtain a working solution. 
      2. After surface staining, dilute and wash cells by adding 150 µl of FACS buffer to each sample.
      3. Centrifuge at 900xg (~2000 rpm) for 3 min to pellet at 4°C. Decant supernatant.
      4. Wash cells by adding 200 µl/well of FACS buffer. Centrifuge as in previous step, then decant. Repeat wash 2x. 
      5. Add 150 µl/well of diluted Fixation/Permeabilization working solution to each tube and resuspend cells thoroughly with pipette. Incubate covered at 4°C for 45 min. 
3. Intracellular Antibody Cocktail Preparation
   1. Prepare a master staining cocktail during the fixation/permeabilization step using the pre-determined appropriate dilutions of each antibody. For each sample, 50 µl of antibody cocktail will be added directly to pelleted PBMC. Calculate volume of each antibody needed and subtract total volume by total antibody volume to determine how much 1X Permeabilization Buffer to add to bring up to final volume.  Be sure to prepare ~20% extra for volume losses. Keep antibody cocktail covered on ice until ready for use. 
   2. See example below for details for a 12-sample staining experiment (12 duplicate stains with specific Ab cocktail + 12 single replicate stains with isotype control cocktail). Scale as necessary based on number of samples and replicate stains, and prepare extra for volume losses.

1.  
2. *Due to the 33x higher concentration of the stock IgG2a-PerCP-eFluor710 isotype control Ab relative to anti-CTLA-4 PerCP-eFluor710 Ab, first pre-dilute IgG2a isotype control Ab by adding 1µl of Ab to 32 µl 1X Perm Buffer.  Mix well, and use this diluted mixture for further diluting 1:50 as above.

1. Preparation of Flow Cytometer Compensation Bead Controls

1. NOTE: This part can be performed concurrently with staining of PBMC samples, during the fix/perm or intracellular staining steps.
2. Prepare by labeling 11 microtiter tubes (1 for each fluorochrome in the panel except for Live/Dead Aqua, plus unstained beads) and add 50 µl of well-mixed UltraComp eBeads (which already includes negative and positive Ab binding beads) to each tube.  For L/D-Aqua staining compensation, add one drop of positive ArC (+) amine reactive beads to 100 µl PBS in a 5-ml polystyrene tube. 
3. Add each antibody at the indicated test volume to its respective single color control tube.  Incubate at 4°C for 10 minutes in the dark (except for Live/Dead Aqua for 30 min incubation) .  Note time of incubation in lab notebook.  Stained UltraComp beads are ready to acquire on the flow cytometer with no further washing or centrifugation.

1.  
2. NOTE: A test is defined as the amount (μg) of antibody that will stain a cell sample in a final volume of 100 μL. If high background is observed on the negative bead population, less antibody can be used. For these cases, it is recommended to use 0.125 ug or less. Because the binding of the antibody to the positive bead is not dependent on the antibody’s specificity, it is not necessary to use the antibody at its optimal concentration. For most antibodies, appropriate compensation values will result when 0.03-1.0 ug of antibody is used in a test.
3. Wash L/D tube by adding 3 mL of FACS buffer.  Centrifuge tube at 400 x g (or ~1500 rpm in Beckman-Coulter Allegra benchtop centrifuge) for 5 minutes. Decant supernatant into sink.  Resuspend in 350 µl FACS buffer and transfer to an appropriately labeled polypropylene microtube. Keep protected from light with aluminum foil and in the dark at 4˚C until acquisition. Add one drop ArC (-) negative beads to the L/D compensation tube immediately prior to acquisition on flow cytometer. 
   1. Intracellular Staining of Fixed and Permeabilized PBMC Samples
      1. After fixation/permeabilization, wash cells by adding 75 µl/well of 1X Permeabilization Buffer.  Centrifuge at 900xg (~2000 rpm) for 3 minutes and decant. Wash again with 225 µl/well of 1X Permeabilization Buffer.  Decant and blot upside down plate on paper towel as described above. 
      2. Add 50 µl/well of prepared intracellular staining antibody cocktail (in 1X Permeabilization Buffer) for each duplicate stain per sample and add 50 µl/well of the intracellular isotype control antibody cocktail to a single replicate of each sample (total of 3 stains per sample in separate wells).  Ensure thorough mixing by pipetting up and down several times.
      3. Cover plate in the dark and leave at 4°C in the refrigerator for exactly 60 min for incubation with antibodies.
      4. Dilute and wash by adding 150 µl/well of 1X Permeabilization Buffer to each sample.
      5. Centrifuge at 900xg (~2000 rpm) for 3 min to pellet at 4°C. Decant supernatant.
      6. Wash cells by resuspending in 200 µl/well of 1X Permeabilization Buffer. Centrifuge as in previous step, then decant. Repeat wash 2x. 
      7. After last decant, resuspend cells in each well in 150 µl FACS Buffer and transfer each to an appropriately labeled 1.2-mL microtiter tube inside an appropriate 96-slot rack for microtiter tubes.  Wash plate wells a second time with 150 µl FACS Buffer and then add to each sample’s appropriate microtiter tube bringing to a final total volume of 300 µl per stain.  
4. NOTE: Ensure that cells in the final resuspension are free of any debris or clumps that may clog the flow cytometer fluidic lines; if necessary, filter cells into specialized 5 ml polystyrene FACS tubes (Corning #352235) through a cell strainer cap before acquisition. 
5. Cover samples in dark with aluminum foil and store at 4˚C until ready for acquisition.  Flow acquisition must be done within 24 hrs.

1. Acquisition of Samples on Flow Cytometer
   1. Using forceps, transfer each microtiter tube into a 5-mL polystyrene FACS tube immediately prior to acquisition and acquire samples on flow cytometer.  Keep samples covered with aluminum foil and cold by placing on ice or using a cool pack until ready to acquire. 
   2. Verify that an experimental template has been created on the acquisition software associated with the flow cytometer that includes date of experiment, date sample was taken, analyst, sample IDs, clinical study protocol ID, sample study timepoint, and any other identifiers as well as antibody specificities for all fluorescence parameters to be assessed. 
   3. Run and save each single-color and unstained compensation control sample with beads and have the flow cytometer calculate correct fluorescence compensation automatically. 
   4. Run all other appropriate control samples (unstained) to verify that voltage settings are correct and that cells are in scale in FSC x SSC plots. 
   5. Set FSC threshold to a value of 5,000-20,000 to exclude debris depending on how FSC voltage settings appear for cell samples. 
   6. For experimental samples, collect by recording 100,000 live cell events for each sample.  If fewer than this number of cells are acquired, these samples should be flagged for low event count. 

1. NOTE: Cells should be acquired at a steady flow rate that does not affect the formation of a single cell stream entering the flow cell; a flow rate that is too high may negatively impact the resolution and consistency of cell staining patterns. 
2. Immediately after experimental sample acquisition run, be sure to clean the fluidic lines of the flow cytometer by running:
   1. 1 FACS tube containing 3 ml BD FACS Clean Solution (contains bleach), 5 min on HI flow rate
   2. 1 FACS tube containing 3 ml BD FACS Rinse (or ddH₂O), 5 min on HI flow rate
   3. 1 FACS tube containing 3 ml BD ddH₂O, 5 min on HI flow rate
3.  
4. Run CST (Cytometer Setup & Tracking) control beads according to manufacturer’s instructions to verify instrument performance (if instrument does not PASS, more extended instrument cleaning is required).  Results should be documented and saved on the computer hard drive. 
5. Place a tube of 1 ml ddH₂O on the sample injection port and place the instrument on STANDBY for next user or shut down the instrument if you are the last person using the instrument for the day.
6.  

1. Tips and troubleshooting
   1. Tip: Incubation time and temperature are crucial for duplication and precision. Use a timer and a temperature-monitored environment for best results. 
   2. Tip: After transferring cells to microtubes, take 350 ml of FACS buffer and serially wash out several (10-15) plate wells of any remaining, fully stained experimental cell samples by pipetting up and down and then moving on to the next well with the same 350 ml.  Dispense the wash volume with the residual cells into an appropriately labeled microcentrifuge tube.  Use this tube of fully stained cells to determine appropriate FSC and SSC voltages and to set gates before acquiring test and QC samples.
   3. Troubleshooting: If CD4 and CD8 populations are bleeding upward (or multiple populations are observed) this could be a result of Qdot particle aggregation.  Be sure to spin down Qdot conjugated antibodies as described above to prevent this issue. 

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