Author
Date
Version
SOP #
Stephanie Swift
26th January 2015
1
H002
Reageants
FACS Buffer (2.5g BSA in 500ml PBS)
Ficoll (or Leukosep)
PBS
Tetramer-APC (project-specific)
CD8-PerCPCy5.5 (BD Biosciences, Clone RPA-T8)
CD3-FITC (BD Biosciences, Clone OKT3)
CD28-PE (BD Biosciences, Clone CD28.2)
CCR7-PECy7 (BD Biosciences, Clone 3D12)
CD45RA-APC-eFluor780 (eBioscience, Clone HI100)
Viability Dye-eF450 (eBioscience # 65-0863-14)
Objective: This protocol addresses the tetramer/immunophenotyping antibody
staining of human PBMCs
H003 - Human Tetramer Immunophenotyping Stain
1. Resuspend freshly-isolated PBMCs at 2x10^7 per ml, and plate 100ul
(2x10^6 cells) per well of a 96-well U-bottom plate.
2. Wash cells with 100ul FACS buffer. Spin down at 1500RPM for 5 mins. at
4C.
3. Prepare master mix of tetramer, remembering to spin tetramer briefly (3 secs)
at 1000RPM before pipetting from the top of the vial. Add 50ul per well for 30
mins. at 4C. See example calculation below for 20 wells:-
Tetramer-APC
FACS Buffer
Dilution
10ul per well
-
Volume (ul)
200
800
4. Wash in 150ul FACS buffer. Spin down at 1500RPM for 5 mins. at 4C.
5. Wash in 200ul FACS buffer. Spin down at 1500RPM for 5 mins. at 4C.
6. Prepare master mix of phenotyping antibodies; add 50ul per well for 30 mins.
at 4C. See example calculation below for 20 wells:-
CD8-PerCPCy5.5
CD3-FITC
CD28-PE
CCR7-PECy7
CD45RA-APC-eFluor780
FACS Buffer
Dilution
1:200
1:200
1:20
1:50
1:50
-
Volume (ul)
5
5
50
20
20
900
7. Wash in 150ul FACS buffer. Spin down at 1500RPM for 5 mins. at 4C.
8. Wash in 200ul FACS buffer. Spin down at 1500RPM for 5 mins. at 4C.
9. Allow vial of viability to dye to warm up to room temperature before opening.
Prepare master mix of viability dye; add 100ul per well for 30 mins. at 4C.
See example calculation below for 20 wells:-
This protocol is shared by the Stojdl Lab under a creative commons license.
Viability Dye-eF450
PBS
Dilution
1:1000
-
Volume (ul)
2
1998
10. Wash in 100ul FACS buffer. Spin down at 1500RPM for 5 mins. at 4C.
11. Wash in 200ul FACS buffer. Spin down at 1500RPM for 5 mins. at 4C.
12. Final resuspension of cells in 200ul FACS buffer. Filter through nylon mesh
just prior to running. Keep cells on ice until running.
This protocol is shared by the Stojdl Lab under a creative commons license.