May 19, 2022
Immunophenotyping for NHPs, containment protocol
- Jonathan Audet1,
- Courtney Meilleur1
- 1Public Health Agency of Canada
Protocol Citation: Jonathan Audet, Courtney Meilleur 2022. Immunophenotyping for NHPs, containment protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v9j2b1g3e/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 31, 2020
Last Modified: May 19, 2022
Protocol Integer ID: 45922
Keywords: flow cytometry, nonhuman primate, immunophenotyping, whole blood, broncho-alveolar lavage, nk cell, cd45ra for memory phenotype, antibody mix, protocol for rhesus macaque, cells from bronchoalveolar lavage, neutrophil, monocyte, cynomolgus macaque, cell, containment lab, eosinophil, containment protocol, basophil
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Abstract
This is a protocol used to perform immunophenotyping of 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 AGM only). It allows the characterization of T cells (CD69/CD25 for activation; CCR7/CD45RA for memory phenotype; α/β vs γ/δ ), B cells, Monocytes, Neutrophils, Basophils, Eosinophils, an potentially NK cells (CD56 does not work for AGM).
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
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
PBS
Human TruStain FcXBioLegendCatalog #422302
BD FACS Lysing Solution (10X)Becton Dickinson (BD)Catalog #349202
BD Cytofix/CytopermBecton Dickinson (BD)Catalog #554722
Ghost Dye Red 780Tonbo BiosciencesCatalog #13-0865-T500
Troubleshooting
Safety warnings
-
Before start
The antibody mix described in the methods was tested on African green monkey whole blood and BAL fluid. CD56 does not stain AGM NK cells. All antibodies should cross-react with cynomolgus and rhesus macaques but the panel might need to be re-titrated.
Preparations
1h 10m
Prepare the staining mix. (for 1 sample:)
| A | B | C | D | E | |
| Supplier | Antibody | Clone | Channel | Volume per test | |
| BD Biosciences | CD45 | D058-1283 | BUV395 | 1.25 | |
| BD Biosciences | CD3 | SP34-2 | BUV496 | 5 | |
| BD Biosciences | CD8 | RPA-T8 | BUV563 | 1.25 | |
| BD Biosciences | CD16 | 3G8 | BUV737 | 5 | |
| BD Biosciences | CD45RA | 5H9 | BV421 | 0.625 | |
| BD Biosciences | CD49d | 9F10 | BV480 | 5 | |
| BD Biosciences | CD4 | L200 | BV605 | 0.625 | |
| BD Biosciences | CD14 | M5E2 | BV650 | 5 | |
| BD Biosciences | CD123 | 7G3 | BV786 | 1.25 | |
| BD Biosciences | CD25 | M-A251 | BB515 | 5 | |
| Miltenyi Biotec | CD66abce | TET2 | PerCP-Vio700 | 1 | |
| BD Biosciences | CD56 | MY31 or NCAM16.2 | BV711 | 5 | |
| BD Biosciences | CD163 | GHI/61 | PE | 20 | |
| BD Biosciences | CCR7 | 2-L1-A | PE-CF594 | 0.625 | |
| BioLegend | HLA-DR | L243 | PE/Fire640 | 1.25 | |
| BD Biosciences | CD69 | FN50 | PE-Cy7 | 5 | |
| BD Biosciences | TCRgd | B1 | APC | 5 | |
| BD Biosciences | CD20 | 2H7 | Alexa 700 | 0.625 | |
| Total | 68.5 | ||||
| BD Brilliant Stain | 31.5 |
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
Prepare a 1:100 dilution of viability dye combining 495 µL PBS and 5 µL Ghost Dye Red 780Tonbo BiosciencesCatalog #13-0865-T500
For whole blood, use the Ghost Dye undiluted.
Surface Staining
30m
Put 5 µl of TruStain FcX in FACS tubes (1 tube per sample).Human TruStain FcXBioLegendCatalog #422302
Equipment
FACS Tube
NAME
Tube
TYPE
Falcon
BRAND
14-959-2A
SKU
Add 100 µl of whole blood (EDTA blood) or BAL cells to the correct tube. Mix.100 µL Sample
Incubate 10 min at Room Temperature (RT).00:10:00 Room temperature
10m
Add 5 µl of the diluted Ghost Dye Red 780Tonbo BiosciencesCatalog #13-0865-T500
If staining whole blood: Add 5 µl of undiluted 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)
Final Wash & Run
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 PBS in FACS tubes.PBS
Spin at 500 x g for 8-10 min.500 x g, 20°C, 00:08:00
Decant supernatant.
Resuspend in 200 µl of PBS.PBS
Run on FACSymphony A5.
Gating strategy for whole blood:
Gating_blood.pdf