Dec 04, 2022
Whole blood T cell assay for NHPs, containment protocol
Forked from Immunophenotyping for NHPs, containment protocol
- Jonathan Audet1,
- Courtney Meilleur1
- 1Public Health Agency of Canada
Protocol Citation: Jonathan Audet, Courtney Meilleur 2022. Whole blood T cell assay for NHPs, containment protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l692prlqe/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: December 02, 2022
Last Modified: December 04, 2022
Protocol Integer ID: 73500
Keywords: flow cytometry, nonhuman primate, whole blood, broncho-alveolar lavage, T cell assay, protocol for rhesus macaque, using whole blood, assay, concentration of the stimulant, antibody mix, cytokine, cynomolgus macaque, antibody, cells from bronchoalveolar lavage, tc1, stimulant, cell, containment lab
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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.
Guidelines
For BAL fluid, make sure to set FSC voltage much lower than for whole blood.
Materials
Human TruStain FcXBioLegendCatalog #422302
BD FACS Lysing Solution (10X)Becton Dickinson (BD)Catalog #349202
BD Cytofix/CytopermBecton Dickinson (BD)Catalog #554722
BD Perm/Wash bufferBecton Dickinson (BD)Catalog #554723
Ghost Dye Red 780Tonbo BiosciencesCatalog #13-0865-T500
Equipment
FACS Tube
NAME
Tube
TYPE
Falcon
BRAND
14-959-2A
SKU
Equipment
2 ml screw-cap tubes
NAME
Microtubes
TYPE
Sarstedt
BRAND
72.694.006
SKU
PBS
Protocol materials
Ghost Dye Red 780Tonbo BiosciencesCatalog #13-0865-T500
PBS
Human TruStain FcXBioLegendCatalog #422302
BD FACS Lysing Solution (10X)Becton Dickinson (BD)Catalog #349202
BD Cytofix/CytopermBecton Dickinson (BD)Catalog #554722
BD Perm/Wash bufferBecton Dickinson (BD)Catalog #554723
Troubleshooting
Safety warnings
-
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.
Stimulation
Aliquot 100 µL whole blood into a tube or well.
Add 200 µL cRPMI 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 5% CO2 Overnight .
Add monensin and brefeldin A, assuming 1e6 cells.
Anti-CD107a BV421 can be added here as well (5 µL )
Mix and incubate at 37 °C 5% CO2 06:00:00 .
6h
Preparations
1h 10m
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.
BD FACS Lysing Solution (10X)Becton Dickinson (BD)Catalog #349202
Surface Staining
30m
Put 5 µl of TruStain FcX in each tube/well.Human TruStain FcXBioLegendCatalog #422302
Incubate 10 min at Room Temperature (RT).00:10:00 Room temperature
10m
Add 5 µl of the Ghost Dye Red 780Tonbo BiosciencesCatalog #13-0865-T500
If staining BAL: Add 5 µl of 1:100 diluted Ghost Dye.
Incubate 20 min at RT in the dark.Room temperature
00:20:00
20m
Add stain mix. Mix.100 µL
Incubate 20 min at RT in the dark.Room temperature
00:20:00
20m
RBC Lysis
Add 2 ml of 1X FACS Lysing solution. Vortex immediately, but gently.2 mL
Incubate no more than 12 min at RT in the dark.00:10:00 No more than 12 minutes
10m
Spin at 300 x g for 5 min.300 x g, 20°C, 00:05:00
Decant supernatant.
Add 2 ml of PBS. Vortex.2 mL
PBS
Spin at 300 x g for 5 min.300 x g, 20°C, 00:05:00
Decant supernatant.
Sample Inactivation
30m
Resuspend in Cytofix/Cytoperm. BD Cytofix/CytopermBecton Dickinson (BD)Catalog #554722
100 µl per 5 x 105 cells.
Use at least 400 µl for easy decanting.
Incubate at least 30 min at RT in the dark.00:30:00 Room temperature In the dark.
30m
Spin at 500 x g for 8-10 min.500 x g, 20°C, 00:08:00
On a clean bench, decant supernatant.
Use same volume of Cytofix/Cytoperm as before to resuspend the cells.BD Cytofix/CytopermBecton Dickinson (BD)Catalog #554722
Transfer in a 2 ml screwcap tube. Shake the tube to cover all surfaces with Cytofix/Cytoperm.
Equipment
2 ml screw-cap tubes
NAME
Microtubes
TYPE
Sarstedt
BRAND
72.694.006
SKU
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
30m
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 BDBD Perm/Wash bufferBecton Dickinson (BD)Catalog #554723 in FACS tubes.
Spin at 500 x g for 8-10 min.500 x g, 20°C, 00:08:00
Decant supernatant.
Repeat steps 31-33.
Prepare the staining mix. (for 1 sample:)
(uses BD Perm/Wash bufferBecton Dickinson (BD)Catalog #554723 )
| 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 00:30:00
30m
Add 1 mL BD Perm/Wash bufferBecton Dickinson (BD)Catalog #554723 .
500 x g, 4°C, 00:08:00
8m
Decant supernatant and blot tubes/plate.
Repeat steps 38-40.
Resuspend in PBS 1% formaldehyde.
Run on flow cytometer.
Gating strategy for whole blood: