Description

This protocol is recommended for the antigenic stimulation of PBMCs in 96-well microplates.  It is based on our work with normal and HIV+ donors using both freshly isolated and cryopreserved cells.  We optimized this protocol with regard to cell recovery and magnitude of cellular response using cytokine flow cytometry as the measurement and antigenic stimulation in tubes as the predicate standard.

Materials and Methods

For a complete list of BD Biosciences CFC products, please refer to our web site www.bdbiosciences.com, or refer to our 2001 catalog. To place an order or to obtain technical support, please call Customer Service at 1-877-232-8995 (toll free) and follow the prompt for flow cytometry instruments and reagents.

Table 1 CFC Staining and Activation Reagents

^a BDIS: BD Biosciences, Immunocytometry Systems

^b BD FastImmune intracellular detection kits include Brefeldin A, EDTA, BD FACS Lysing Solution, and BD FACS                Permeabilization Solution 2.

^c No specific manufacturer recommended

Table 2 Accessory Products and Instrumentation

^a BDIS: BD Biosciences, Immunocytometry Systems.  BDDL: BD Biosciences, Discovery Labware

^b No specific manufacturer recommended

Please follow all recommended precautions that are provided in the technical data sheet of each manufacturer’s product.

Instructions for Processing Reagents

Antigens

SEB (positive activation control): Add 2 mL of sterile PBS directly to a 1-mg vial of SEB.  Cap the vial and shake to dissolve all the powder.  Store this stock solution at 4^oC.  On day of use, prepare working stock by diluting 1:10 in sterile PBS.

Peptides: Dissolve single peptides in DMSO at a concentration of 2 mg/mL.  Freeze aliquots of 5 µL each (for stimulation of 1 mL of PBMCs at a final concentration of 10 µg /mL) at ^–80^oC.  On day of use, prepare working stock by diluting 1:10 in sterile PBS.

Peptide mix (e.g. CMV pp65 or HIV Gag p55): Please refer to the BD Biosciences, Immunocytometry Systems protocol Preparation and Use of Peptide Mixes.  On day of use, dilute 1:10 in sterile PBS to arrive at a working stock of 0.07 mg/mL per peptide.

Brefeldin A (BFA) from FastImmune kit

Upon receipt, thaw BFA, dispense into 10 ml aliquots, and store at ^–20^oC.  On day of use, remove an aliquot from the freezer, and dilute 1:10 with sterile PBS.  Discard any unused portion.

BD FACS Lysing Solution and BD FACS Permeabilizing Solution 2 from FastImmune kit

Dilute each 10X solution in deionized water to make 1X working solution.  Store at room temperature.

BD CD3 APC

Prior to use, dilute bottled reagent 1:16 in wash buffer, as a drop-in addition to the FastImmune CD4 Intracellular Detection kits.  Use 5 ml per test.  

Paraformaldehyde in PBS, 1%

Dilute 10% solution of paraformaldehyde 1:10 in 1X PBS.  Store at 4^oC. 

Wash buffer

First prepare stock solutions of 5% BSA in deionized water (filter sterilize) and 10% NaN₃ in deionized water.  Then prepare 500 mL of wash buffer by adding 50 mL of 5% BSA stock solution and 5 mL of 10% NaN₃ stock solution to 445 mL of 1X sterile PBS.  This represents final concentrations of 0.5% BSA and 0.1% NaN₃ in PBS. Store at 4^oC.

Complete RPMI (cRPMI)

Supplement sterile RPMI-1640 medium with 10% sterile heat-inactivated FBS and 1% sterile antibiotic/antimycotic.  Store at 4^oC.

Protocol

Preparation

Fresh PBMCs

Prepare a working concentration of 5x10⁶/mL in room temperature cRPMI. Proceed to "Stimulation" directions, below.

Cryopreserved PBMCs

1.        Follow "Thawing Procedure" section of the BD Biosciences, Immunocytometry Systems protocol Freezing and Thawing Procedures for PBMC. 

2.        Resuspend at a final working concentration of 5x10⁶ PBMCs/mL in room temperature cRPMI media. 

3.        Check for clumps and remove them with a pipettor tip.

4.        Plate 200 ml/well in a round-bottom 96-well plate.  This will result in 1x10⁶ cells per well (we have tested 5x10⁵ to 2x10⁶ cells per well with equivalent results).  Prepare additional wells for manual compensation, if desired.

5.        Incubate covered plate at 37°C for 12–18 hours.

 

Stimulation

1.        Prepare working stock solutions of activating reagents.

2.        Label three tubes as "NS" (for non-stimulated control), "SEB," or "Peptide."  Prepare stimulation reagents in bulk by combining costimulatory mAbs CD28+CD49d, stimulus (add nothing to non-stimulated control), BFA, and PBS into the appropriately labeled tubes.  Add appropriate amounts such that costim + stimulus + BFA + PBS = 20 mL for each condition on the plate, times the number of wells used (plus at least one extra well to account for fluid loss). 

Stimulus preparation per well:

3.        Add 200 ml of PBMCs, either fresh or those rested overnight in incubator.  Mix well by pipetting.

4.        Dispense 20 mL of appropriate stimulus reagent to each well of a 96-well round bottom plate.

5.        Incubate covered plate for six hours at 37°C.

6.        Add 20 mL of EDTA to each well and incubate for 15 minutes at room temperature.  

7.        Mix cell suspension well with multichannel pipette.

8.        Centrifuge plate at 250 x g for five minutes; aspirate supernatant with manifold.  Approximately 30 ml will remain in wells.

9.        Resuspend cells in 100 mL 1X BD FACS Lysing Solution.  Incubate at room temperature for 10 minutes.

Permeabilization and Staining

1.        Add 100 mL cold wash buffer to each well, and centrifuge at 500 x g for five minutes.

2.        Aspirate supernatant and resuspend cells in 200 mL 1X BD FACS Permeabilizing Solution 2. Incubate at room temperature for 10 minutes. 

3.        Centrifuge at 500 x g for five minutes. Aspirate supernatant.

4.        Resuspend cells in 200 mL cold wash buffer and centrifuge at 500 x g for five minutes. Aspirate supernatant.  

5.        Add staining mAbs to each well, and mix by pipetting.  Add single-color mAbs to compensation control wells, if used.

6.        Incubate plate at room temperature for 30-60 minutes in the dark. 

7.        Add 175 mL of cold wash buffer and centrifuge at 500 x g for five minutes; aspirate supernatant.

8.        Repeat wash, centrifugation, and aspiration with 200 mL of wash buffer. 

9.        Resuspend pellet with 200 mL cold 1% paraformaldehyde. 

10.     Keep plate at 4°C in the dark until FACS acquisition, which should be performed within 2-18 hours. 

Acquisition

1.        Using BD FACSComp™ software and BD CaliBRITE™ reagents, set compensation on BD FACSCalibur.

2.        Make sure cells are well suspended before acquisition.

3.        Acquire at least 10,000 relevant (CD4+ or CD8+) events, preferably 40,000.  For best results, determine optimal sample size with the BD Biosciences, Immunocytometry Systems reference spreadsheet Calculating Sample Size for Rare Event Analysis.