Whole blood T cell assay for NHPs, containment protocol
Jonathan Audet, Courtney Meilleur
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Abstract
This is a protocol used to perform T cell assays using whole blood (collected in EDTA tubes) or cells from bronchoalveolar lavage (BAL). We have successfully used this protocol for rhesus macaques, cynomolgus macaques, and African green monkeys (although antibody mix provided was titrated on macaques). It allows the characterization of T cells into Th1 and Th2 (or Tc1 and Tc2) phenotypes. It technically offers 64 different activation profiles (4 cytokines + CD107a + CD69).
The nature and concentration of the stimulant is left unspecified as we have used different ones depending on reagent availability.
This protocol was designed to deal with samples coming from containment labs (> CL2) at our facility. If you are using this protocol at a different facility please ensure that proper testing and approvals are in place. The protocol can be used for experiments completed entirely in CL2, simply go from step 17 directly to 31.
Before start
The antibody mix described in the methods was tested on Cynomolgus macaque whole blood and BAL fluid. All antibodies should cross-react with rhesus macaques and African green monkeys but the panel might need to be re-titrated.
Steps
Stimulation
Aliquot 100µL into a tube or well.
Add 200µL containing 1.5 X the desired concentration of stimulant.
(Stimulant(s) and concentration depend on the experiment and can include anti-CD28 and anti-CD49d)
Mix and incubate at 37°C .
Add monensin and brefeldin A, assuming 1e6 cells.
Anti-CD107a BV421 can be added here as well (5µL )
Mix and incubate at 37°C 6h 0m 0s .
Preparations
Prepare the staining mix. (for 1 sample:)
| A | B | C | D | E |
|---|---|---|---|---|
| Supplier | Antibody | Clone | Channel | Volume per test |
| BD Biosciences | CD3 | SP34-2 | Alexa Fluor 700 | 5 |
| BD Biosciences | CD8 | RPA-T8 | BV786 | 5 |
| BD Biosciences | CD45RA | 5H9 | PE-CF594 | 2.5 |
| BD Biosciences | CD4 | L200 | PerCP-Cy5.5 | 20 |
| BD Biosciences | CCR7 | G043H7 | BV711 | 5 |
| BD Biosciences | CD69 | FN50 | BV650 | 5 |
| Total | 42.5 | |||
| BD Brilliant Stain | 57.5 | |||
Prepare the 1X FACS Lysing solution by diluting the 10X stock with Milli-Q water. You will need 2 ml of 1X solution for each sample.
Surface Staining
Put 5 µl of TruStain FcX in each tube/well.
Incubate 10 min at Room Temperature (RT).0h 10m 0s Room temperature
Add 5 µl of the
If staining BAL: Add 5 µl of 1:100 diluted Ghost Dye.
Incubate 20 min at RT in the dark.20Room temperature
0h 20m 0s
Add stain mix. Mix.100µL
Incubate 20 min at RT in the dark.20Room temperature
0h 20m 0s
RBC Lysis
Add 2 ml of 1X FACS Lysing solution. Vortex immediately, but gently.2mL
Incubate no more than 12 min at RT in the dark.0h 10m 0s
Spin at 300 x g for 5 min.300x g,20°C
Decant supernatant.
Add 2 ml of PBS. Vortex.2mL
Spin at 300 x g for 5 min.300x g,20°C
Decant supernatant.
Sample Inactivation
Resuspend in Cytofix/Cytoperm.
100 µl per 5 x 105cells.
Use at least 400 µl for easy decanting.
Incubate at least 30 min at RT in the dark.0h 30m 0s Room temperature
Spin at 500 x g for 8-10 min.500x g,20°C
On a clean bench, decant supernatant.
Use same volume of Cytofix/Cytoperm as before to resuspend the cells.
Transfer in a 2 ml screwcap tube. Shake the tube to cover all surfaces with Cytofix/Cytoperm.
Equipment
| Value | Label |
|---|---|
| 2 ml screw-cap tubes | NAME |
| Microtubes | TYPE |
| Sarstedt | BRAND |
| 72.694.006 | SKU |
| https://www.sarstedt.com/en/ | LINK |
Transfer tubes from containment space to CL2 space according to the facility's approved protocols/SOPs.
Tubes can be opened in CL2 (in a BSC) no less than 30 min after the resuspension (step 17).
(Samples are generally processed the next day; keep at 4 C overnight, in the dark)
Intracellular staining
Give tubes a quick spin in a tabletop centrifuge.
Ensure all tubes have at least a few hundred microliters of Cytofix/Cytoperm.
Transfer the samples into 1 ml of BD
Spin at 500 x g for 8-10 min.500x g,20°C
Decant supernatant.
Repeat steps 31-33.
Prepare the staining mix. (for 1 sample:)
(uses
| A | B | C | D | E |
|---|---|---|---|---|
| Supplier | Antibody | Clone | Channel | Volume per test |
| BioLegend | IFNgamma | B27 | APC | 2.5 |
| BioLegend | TNF | MAb11 | PE-Cy7 | 5 |
| BioLegend | IL-2 | MQ1-17H12 | Alexa Fluor 488 | 0.625 |
| BioLegend | IL-4 | 8D4-8 | PE | 2.5 |
| Total | 10.625 | |||
| PermWash | 89.375 |
Resuspend in 100 µl of intracellular staining mix.
Incubate 4°C 0h 30m 0s
Add 1mL
500x g,4°C
Decant supernatant and blot tubes/plate.
Repeat steps 38-40.
Resuspend in PBS 1% formaldehyde.
Run on flow cytometer.
Gating strategy for whole blood:
