Isolate Peripheral Blood Mononuclear Cells (PBMC)
Prepare PBMC by density gradient centrifugation.
Antibody staining for cell sorting
The following procedure describes how to stain the controls and cell sample for sorting. The kit does not require the use of isotype controls to perform a successful sort. Each population has been well characterized and verified for staining patterns. However, you may wish to use your own fluorescence minus one (FMO) isotype controls as desired.
Note: Follow aseptic stain and sort procedures if planning to expand isolated Treg population.
1. Label five 12 × 75-mm tubes for compensation. Follow instructions from Cat. No. 552843 Anti-Mouse Ig, κ/Negative Control (FBS) Compensation Particles Set. Prepare beads for flow cytometry per the recommended assay procedure.
Tube 1: Unstained Compensation beads
Tube 2: FITC Compensation beads
Tube 3: PE Compensation beads
Tube 4: PerCP-Cy5.5 Compensation beads
Tube 5: AlexaFluor® 647 Compensation beads
Tube 6: Optional: Horizon V450 compensation tube for post sort FoxP3 staining
2. Add the following antibodies from the single vials contained in the kit to the tubes labeled in Step #1.
-FITC Compensation tube: 20 µl of CD45RA FITC
-PE Compensation tube: 20 µl of CD25 PE
-PerCP-Cy5.5 Compensation tube: 20 µl of CD4 PerCP-Cy5.5
-AlexaFluor® 647 Compensation tube: 20 µl of CD127 APC
Prepare beads for flow cytometry as described in the instructions for Cat. No. 552843, Anti-Mouse Ig, κ/Negative Control (FBS) Compensation Particles Set.
3. Stain Sample: Aliquot the sample to be sorted into a 50 ml tube for staining the PBMC or CD4 enriched cells. For total PBMC numbers less than 100 million the staining concentration is 20 million per 1.0 ml. For total PBMC numbers greater than 100 million, the staining concentration is 100 million per 1.0 ml.
Note: Test size equals 200 µl per 100 million cells. The wash buffer is 1x PBS + 1% Human AB serum.
4. Add antibody: 200 µl each of Cocktail and CD45RA per 100 million cells. For example:
-120 million cells, add 240 µl of Cocktail and CD45RA to a final volume of 1.2 ml
- 50 million cells, add 100 µl of Cocktail and CD45RA to a final volume of 2.5 ml
5. Incubate protected from light at 18°C - 22°C for 30 min.
6. Add enough wash buffer (e.g. 49 ml) to fill 50 ml tube and centrifuge at 250 x g for 15 min.
7. Aspirate the supernatant and resuspend the sample at a concentration of 5 - 10 million cells/ml in wash buffer.
8. Prepare reception tubes. Coat 12 × 75-mm polypropylene tubes with 0.2 ml of human AB serum, invert, then add 0.2 ml of media [(formulation; X-VIVO-15 (Cambrex) + 10% Human AB serum (Sigma) + 0.2% Acetylcysteine solution)] or use PBS wash buffer.
9. Use the FACSAria™ cell sorter to isolate the cell populations of interest.
Population options for sorting:
Core population CD4+/CD25int-hi/CD127lo
10. Collect fractions in 12 × 75-mm polypropylene tubes coated with human AB serum, and containing media.
11. Centrifuge fractions at 250 x g for 10 minutes, remove supernatant without disturbing the pellet. Resuspend in wash buffer and recover at least 30,000 cells for FoxP3 staining or post sort acquisitions, and/or at least 50,000 cells for expansion.
Note: be sure to use 1x PBS + 1% Human AB serum when acquiring post sort fractions.
12. Proceed with modified FoxP3 staining protocol (below), expansion, suppression, or other downstream protocol of choice.
Sorting Protocol. A 70 or 100 micron nozzle may be used for this procedure. A 70 micron nozzle will allow for flow rates and pressures high enough to sort ~100 million PBMC in 0.5-2.5 hours to obtain ~90,000 CD45RA+ T regulatory cells depending upon the donor. A 100 micron nozzle may allow for higher purities and viabilities while approximately doubling the sort time. Sorting of 200 million cells or more may be required for adequate recoveries of CD25brightCD45RA- T cells. See figure and figure legend for complete gating guidelines.
FoxP3 post sort staining protocol. The following procedure describes a modified FoxP3 staining protocol for post sort fractions. Handle cells minimally and avoid high g forces after sorting. A successful sort of 100-250 million PBMC will provide enough cells to test for viability and FoxP3 expression in a verification experiment and likely provide enough cells for expansion. However, we recommend conserving CD45RA fractions for purity measurements and stain cells for FoxP3 when cells are plentiful later in expansion. We recommend staining in small polypropylene tubes or plates. Surface staining is not required if post sort fractions are fixed and tested the day of the sort, but FITC fluorescence signal to noise will be reduced by this procedure. Alternatively, costain expanded cells with CD3 (Cat. No. 555333) and/or CD4 (Cat. No. 565999), Human Regulatory T Cell Cocktail (Cat. No. 560249) or stain with 10 µl of this kit per test.
1. Bring the buffers to RT before use. Prepare working solutions of the BD Pharmingen Human FoxP3 Buffer Set
(Cat. No. 560098) and follow this modified protocol:
2. Label five polypropylene 0.65 ml microcentrifuge tubes (VWR MN 87003-290)
Tube 1: Pre sort fraction
Tube 2: CD4+CD25++CD127lowCD45RA- Horizon V450 isotype control (Cat. No. 560373)
Tube 3: CD4+CD25++CD127lowCD45RA- Horizon V450 FoxP3 (Cat. No. 560460)
Tube 4: CD4+CD25++CD127lowCD45RA+ Horizon V450 isotype control
Tube 5: CD4+CD25++CD127lowCD45RA+ Horizon V450 FoxP3
3. Prepare human sorted fractions for FoxP3 staining.
4. Centrifuge the receiving tubes after the sort 250 x g for 10 minutes, gently remove wash buffer.
5. Dilute cells to at least 30,000 cells per 50 µl in BD Pharmingen Stain Buffer (FBS).*
Note: use 200,000 cells for Tube 1 to assist in adjusting FSC and SSC when acquiring fixed cells.
6. Add 500 µl of BD Pharmingen Stain Buffer (FBS). Centrifuge 250 x g for 5 minutes, gently remove wash buffer.
7. To fix cells, add 250 µl of 1x Buffer A per tube, vortex gently for one second, incubate for 10 minutes at RT protected from light.
Note: Optional step, store overnight at 4°C and continue procedure the next day. Bring to RT before step 8.
8. Centrifuge 250 x g for 5 minutes, and remove fixative. Caution: Be aware the pellet is buoyant.
9. To permeabilize the cells, gently re-suspend pellet in residual volume fix buffer and then add 250 µl of a 1x working solution of Human FoxP3 Buffer C to each tube. Vortex gently for one second, incubate for 30 minutes at RT protected from light.
10. Add 500 µl of BD Pharmingen Stain Buffer (FBS). Centrifuge 250 x g for 5 minutes, gently remove wash buffer leaving behind ~50 µl of residual buffer.
11. Add V450 conjugated FoxP3 antibody and/or isotype control at 0.5x test size indicated on the vial. Vortex gently for one second.
12. Incubate for 30 minutes in the dark at RT.
13. Repeat wash step #9.
14. Resuspend in 300 µl 1x PBS and analyze immediately. Acquire 500 to 2,000 CD4 positive cell events for best results.
Note: Optimize FSC vs. SSC settings with pre-sort fraction. Carryover from tube to tube can affect interpretation of data collected on very small fractions of cells. It is advised to acquire a tube of PBS between runs to define background and/or carryover from previous tubes in the run.
* We recommend using the BD Pharmingen Stain Buffer (FBS; Cat No. 554656) for initial surface staining and all wash steps and covering tubes during incubation steps with caps or parafilm. We also recommend optimizing forward scatter and side scatter voltages to visualize lymphocytes as separate from debris and/or platelets before acquisition.
BD® CompBeads can be used as surrogates to assess fluorescence spillover (compensation). When fluorochrome conjugated antibodies are bound to BD® CompBeads, they have spectral properties very similar to cells. However, for some fluorochromes there can be small differences in spectral emissions compared to cells, resulting in spillover values that differ when compared to biological controls. It is strongly recommended that when using a reagent for the first time, users compare the spillover on cell and BD® CompBeads to ensure that BD® CompBeads are appropriate for your specific cellular application.