Author
Date
Version
SOP #
Stephanie Swift
16th July 2015
4
M004
Objective: This protocol addresses the processing, peptide stimulation and antibody
staining of fresh mouse peripheral blood for flow cytometric analysis
M004 - Intracellular Cytokine Staining
Required reageants and equipment:
ACK lysis buffer (see Appendix A)
1x heparin (see Appendix A) (Sigma #H3149)
5% FCS (25ml FCS + 475ml PBS)
FACS Buffer (2.5g BSA in 500ml PBS)
cRPMI (RPMI + 10% FCS + L-Glut + P/S + HEPES)
Golgi Plug (BD Biosciences #554715)
Cytofix-Cytoperm (BD Biosciences #554715)
Permwash buffer (BD Biosciences #554715; 50ml stock in 450ml dH2O)
CD8-PerCPCy5.5 (BD Biosciences clone 53-6.7)
IFNg-APC (BD Biosciences clone XMG1.2)
TNFa-FITC (BD Biosciences clone MP6-XT22)
IL-2-PE (BD Biosciences clone JES6-5H4)
Viability Dye-eF450 (eBiosciences #65-0863-14)
96-well U-bottom plates
Phorbol 12-myristate 13-acetate (PMA) (Sigma #P-1585)
Ionomycin (Sigma #I-0634)
One Comp eBeads (eBioscience #01-1111-42)
PBS
A. Processing Blood Samples
Blood samples are collected from a non-terminal saphenous bleed (~5 drops per
mouse into a tube containing 200ul 1 x sterile heparin). Always collect blood from two
extra naive mice for your negative (no peptide) and positive (PMA/ionomycin)
controls (plus unstained compensation control).
1. Measure the total volume of blood (remember to subtract the 200ul 1 x
heparin collection solution) collected for each individual mouse, and note
down in labbook.
2. Add 2ml ACK to each tube, flick to resuspend and incubate 5 mins. at RT.
3. Add 6ml PBS to wash. Spin down at 1500RPM for 5 mins. at 4C.
4. Pour off supernatant, resuspend by flicking.
Note. If the pellet is larger than 2mm, proceed on to a second ACK lysis (step
5). Otherwise, proceed straight on to the cRPMI wash in step 7.
5. and add 1ml ACK. Flick to resuspend and incubate 3 mins. at RT.
6. Add 6ml PBS to wash. Spin down at 1500RPM for 5 mins. at 4C.
7. Pour off supernatant, add 2ml cRPMI to wash. Spin down at 1500RPM for 5
mins. at 4C, remove supernatant & resuspend by flicking.
8. Resuspend in a volume of cRPMI that allows you to plate 100ul per well of a
96-well U-bottom plate, taking into account that there will be ~ 50ul left in the
tube after decanting the final cRPMI wash. Ideally, plate 100ul for an
unstimulated control and 100ul for a stimulated control per mouse.
This protocol is shared by the Stojdl Lab under a creative commons license.
Note. If your pellets were small, or you have to split your non-terminal blood
sample into more than two wells, use 50ul for your unstimulated well, or don't
include an unstimulated and simply stimulate blood from a naive mouse with
your peptide(s) of interest as your control. Try to limit splitting of non-terminal
bleed samples to no more than 4 aliquots due to low cell numbers.
B. Peptide Stimulation
9. Add 50ul/well of peptide(s), diluted in cRPMI, to each sample. See Appendix
B for example peptides. e.g. for SIINKFEKL, final concn in well = 1ug/ml.
Stock = 1mg/ml (0.2ul/well).
For negative (unstimulated) controls, add 50ul/well of DMSO diluted in
cRPMI at the highest concentration used in your test peptide stimulations
(see Appendix B).
For positive controls, add 50ul/well of PMA/ionomycin (see Appendix B for
dilutions).
Note. For weak peptides, further stimulation may be necessary with antiCD28 - 0.1ul per sample; reduce cRPMI volumes accordingly.
10. Incubate for 1h at 37C.
11. After 1h, add 50ul diluted Golgi Plug to both unstimulated and stimulated
wells. See below for example calculation for 20 wells:- (per sample, always
make extra to account for pipetting loss/error)
Reageant
Golgi Plug
cRPMI
Dilution
0.2ul per well
-
Volume (ul)
4
996
12. Incubate for a further 4h at 37C.
13. Spin down at 1500RPM for 5 mins. at 4C.
If you wish to leave cells overnight at this point, resuspend cells in 200ul 5%
FCS. Store at 4C o/n.
If you wish to continue immediately to staining, wash cells twice in 200ul
FACS buffer.
C. Intracellular Cytokine Staining (ICS)
Note: The dilutions of each antibody may change between different lots of the same
antibody clone received from the same company. Individual lot titrations should be
carried out to optimise dilutions.
14. Spin down plate and tip off supernatant.
15. Add 50ul diluted Fc block per well, mix and incubate for 15 mins. on ice. See
below for example calculation for 20 wells:- (per sample, always make extra
to account for pipetting loss/error)
Reageant
Fc block
FACS Buffer
Dilution
1:100
-
Volume (ul)
10
990
16. Add 150ul FACS buffer to wash, spin down at 1500RPM for 5 mins. at 4C,
and tip off supernatant.
17. Add 50ul/well of diluted CD8 and/or CD4 antibody together with live/dead
viability stain, mix and incubate for 30 mins. on ice IN THE DARK. See below
This protocol is shared by the Stojdl Lab under a creative commons license.
for example calculation for 20 wells:- (per sample, always make extra to
account for pipetting loss/error)
Reageant
CD8-FITC
Viability Dye-eF450
FACS Buffer
Dilution
1:400
0.1ul per well
-
Volume (ul)
2.5
2.0
995.5
18. Wash 150ul FACS buffer, spin down at 1500RPM for 5 mins. at 4C, and tip
off supernatant.
19. Wash 200ul FACS buffer, spin down at 1500RPM for 5 mins. at 4C, and tip
off supernatant.
20. Fix with 100ul Cytofix-Cytoperm, mix well and incubate for 20 mins. on ice IN
THE DARK.
21. Wash in 100ul permwash buffer, spin down at 1500RPM for 5 mins. at 4C,
and tip off supernatant.
22. Wash in 200ul permwash buffer, spin down at 1500RPM for 5 mins. at 4C,
and tip off supernant. Pellets should now be transparent.
23. Add 50ul diluted intracellular antibody cocktail for 30 mins. on ice IN THE
DARK. See below for example calculation for 20 wells:- (per sample, always
make extra to account for pipetting loss/error)
Reageant
IFNg-APC
TNFa-PECy7
IL-2-PE
Permwash buffer
Dilution
1:100
1:200
1:100
-
Volume (ul)
10
5
10
975
24. Add 150ul permwash buffer, spin down at 1500RPM for 5 mins. at 4C, and
tip off supernatant.
25. Add 200ul permwash buffer, spin down at 1500RPM for 5 mins. at 4C, and
tip off supernatant.
26. Final resuspension in 200ul FACS buffer. Keep cells on ice and in the dark
until running on flow cytometer.
Note: for quantitative analysis (relative #), #’s obtained from FlowJo analysis
will have to be adjusted to account for differences in the total volume in each
blood sample prior to processing. See appendix C.
To prepare compensation controls:1. Vortex compensation beads (eBioscience One Comp eBeads #01-1111-42).
Label a separate tube for each antibody/fluorophore used in your experiment.
2. Add 1 drop beads per tube.
3. Add 1ul of antibody stock. Vortex. Incubate at RT for 15-30 mins. IN DARK.
4. Add 2ml FACS buffer, pellet by centrifugation at 1500RPM for 5 mins. Pour
off supernatant.
5. Resuspend bead pellet in 300ul FACS buffer. Vortex.
This protocol is shared by the Stojdl Lab under a creative commons license.
APPENDIX A
ACK Lysis Buffer
8.29g Ammonium Chloride
1g Potassium Bicarbonate
200ul 0.5M EDTA pH8.0
800ml dH2O
pH to 7.4, then make up to 1L with dH2O
10x Sterile Heparin (1x working stock)
The 10x sterile heparin stock solution should be at 400U/ml.
e.g. weigh 122mg heparin sodium salt (from porcine intestinal mucosa) at 164 USP
units/mg. r/s in 50ml PBS = 400U/ml solution.
Filter stock solution through 0.45um filters. This can be stored for several months at
4 degrees Celsius.
The working stock (to collect blood into) should be diluted 10-times to 40U/ml in
PBS. Can store the working stock for several months at 4 degrees Celsius.
This protocol is shared by the Stojdl Lab under a creative commons license.
APPENDIX B
Preparing Negative Control Master Mix
Add 50ul/well of DMSO diluted in cRPMI at the highest concentration used in your
test peptide stimulations e.g. if you stimulated some wells with 1ul SIINFEKL peptide
in 5 ml cRPMI, and some wells with 10uL m38 peptide in 5 ml, make up your
negative control by adding 10ul of DMSO to 5ml cRPMI.
Preparing Positive Control PMA/Ionomycin Master Mix
1. Phorbol 12-myristate 13-acetate (PMA) powder (Sigma #P-1585)
a. Reconstitute in DMSO at 0.1 mg/ml.
b. Store small aliquots (eg, 20 uL) at –20°C; do not refreeze aliquots after thawing.
c. Dilute 1:100 in sterile PBS (without sodium azide) for each experiment to
prepare a fresh stock solution.
2. Ionomycin powder (Sigma #I-0634)
a. Reconstitute in EtOH at 0.5 mg/ml.
b. Store at –20°C.
c. Dilute 1:10 in sterile PBS (without sodium azide) for each experiment to prepare
a fresh stock solution.
3. Mix fresh PMA solution at a final concentration of 10 ng/ml of cell suspension
(0.5ul per 50ul - in cRPMI) with fresh ionomycin solution at a final concentration of
1ug/mL of cell suspension (1ul per 50ul - in cRPMI). See example calculation below
for 20 wells:Reageant
PMA (1:100 fresh)
Ionomycin (1:10 fresh)
cRPMI
Volume (ul)
10
20
970
Preparing Test Peptide Master Mix
Check stock concentration of peptides – you may need to adjust the following
calculations for different peptides. Default final peptide concentrations are
1ug/ml.
CD8+ Epitopes in H-2b haplotype (i.e. C57BL/6):
Peptide
MHC
H2-Kb
H2-Db
H2-Db
H2-Kb
H2-Kb
H2-Kb
H2-Kb
H2-Kb
H2-Kb
Protein
hDCT180-188
gp10025-33
gp10025-33
OVA257-264
βgal97-104
VSV N52-59
FMT N301-309
MCMV m38316-323
AdV DBP418-426
Peptide
Sequence
SVYDFFVWL
KVPRNQDWL
EGSRNQDWL
SIINFEKL
DAPIYTNV
RGYVYQGL
AVVLMFAQC
SSPPMFRV
FALSNAEDL
Stock
Final
Concentration
Concentration
5mg/ml
10mg/ml
10mg/ml
5mg/ml
5mg/ml
10mg/ml
2ug/ml
1ug/ml
1ug/ml
1ug/ml
1ug/ml
1ug/ml
1ug/ml
10ug/ml
20ug/ml
CD8+ Epitopes in H-2d Haplotype (i.e. Balb/C):
Peptide
MHC
Protein
Peptide
Sequence
Stock
Final
Concentration
Concentration
This protocol is shared by the Stojdl Lab under a creative commons license.
CT26 Tumour Cells
H2-Dd
VSV N275-283
SPSYVYHQF
MPYLIDFGL
1ug/ml
1ug/ml
This protocol is shared by the Stojdl Lab under a creative commons license.
APPENDIX C
Converting Frequencies to Absolute Numbers
If the volume of blood collected from each mouse is known, PBS-treated control mice
were included in the experiment and samples were processed in parallel and run
through the flow cytometer in the same final volume for exactly the same length of
time, then frequencies can be converted to physiologically-relevant absolute #’s as
follows:
1. Add up the total # of lymphocyte events collected on the flow cytometer.
Calculate the mean # of lymphocyte events collected from the PBS control group.
2. Calculate the adjustment factor that converts the mean # of lymphocyte events to
the expected physiological # (=7.07x103/μl blood; Charles River Laboratories
dataset for female C57BL/6 mice; BALB/c=6.69x103/μl blood). Multiply all raw
lymphocyte numbers by this adjustment factor.
3. All lymphocyte subset frequencies can then be multiplied by the adjusted
lymphocyte # to determine their respective #’s.
To estimate total # of cells circulating in the mouse (i.e. scale up from your nonterminal blood volume to the total blood volume of the mouse), assume each mouse
has a total blood volume of 1.89 ml (based on the weight chart for female C57BL/6
mice provided by Charles River Labs and “Methods of blood collection in the mouse”
J. Hoff, Lab Animal, 29:10, pp. 47-53; assumptions: an average age of 10 weeks at
the time of sampling, a mean body weight of 21 g and a mean blood volume of 9 %
of body weight).
This protocol is shared by the Stojdl Lab under a creative commons license.
APPENDIX D - Cell Phenotype Markers
T Cells
Myeloid Dendritic Cells
CD3+
CD4+ and/or CD8+
CD11b+
CD11chi
Regulatory T Cells
B220-
CD3+
CD4+
Gr-1NK1.1-
Foxp3+
CD3-
CD25+
MHC class II+
CTLA4+
Macrophages/Monocytes
GITR+
CD127lo
Gr-1CD11blo/mid
B Cells
CD11c+
B220+
CD19+
B220F40/80+
CD3-
NK1.1-
NK Cells
CD3-
CD3-
MHC class II+
NK1.1/DX5+
Myeloid Suppressor Cells
Plasmacytoid Dendritic Cells
Gr-1+
CD11blo/mid
CD11cmid
CD11b+
CD80+
B220+
CD40-
Gr-1+
B220-
NK1.1-
CD86-
IKDC’s
CD3MHC class
CD19-
II+
B220+
CD11c+
NK T Cells
MHC class II+
CD3+
NK1.1/DX5+
NK1.1/DX5+
CD3Gr-1-
This protocol is shared by the Stojdl Lab under a creative commons license.