Immunophenotyping for NHPs, containment protocol
Jonathan Audet, Courtney Meilleur
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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.
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.
Steps
Preparations
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.
Prepare a 1:100 dilution of viability dye combining 495µL and 5µL
For whole blood, use the Ghost Dye undiluted.
Surface Staining
Put 5 µl of TruStain FcX in FACS tubes (1 tube per sample).
Equipment
| Value | Label |
|---|---|
| FACS Tube | NAME |
| Tube | TYPE |
| Falcon | BRAND |
| 14-959-2A | SKU |
| https://www.fishersci.com/us/en/home.html | LINK |
Add 100 µl of whole blood (EDTA blood) or BAL cells to the correct tube. Mix.100µL
Incubate 10 min at Room Temperature (RT).0h 10m 0s Room temperature
Add 5 µl of the diluted
If staining whole blood: Add 5 µl of undiluted 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)
Final Wash & Run
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.
Spin at 500 x g for 8-10 min.500x g,20°C
Decant supernatant.
Resuspend in 200 µl of PBS.
Run on FACSymphony A5.
Gating strategy for whole blood:
