Escolar Documentos
Profissional Documentos
Cultura Documentos
2.3.
2.4.
2.5.
2.6.
2.7.
2.8.
2.9.
2.10.
2.11.
2.12.
2.13.
2.14.
2.15.
2.16.
2.17.
2.18.
2.19.
2.20.
Biosafety cabinet
2.21.
Centrifuge
2.22.
2.23.
Calibrated pipettes
3. Procedure
Thaw PBMC
3.1.
Warm media to 37C in water bath. Each sample will require 22ml
of media with benzonase. Calculate the amount needed to thaw all
samples, and prepare a separate aliquot of warm media with 1:10000
benzonase (25 U/ml). Thaw no more than 3 samples at a time. Run one
control PBMC with each batch of samples.
3.2.
3.3.
3.4.
3.5.
3.6.
3.7.
3.8.
3.9.
3.10.
3.11.
3.12.
3.13.
3.14.
3.15.
3.16.
Stimulate Cells
3.17.
Stock Concentration
1 mg/mL in
Intermediate Dilution
1:1000 in PBS
Final Concentration
10 ng/ml
myristate13acetate (PMA)
Phytohemagglutinin (PHA)
1:10 in PBS
1 ug/ ml
SEB
(store at 4C)
50 g/mL in PBS
None
1 g/mL (1:50)
Peptide mixes
1:10 in PBS
1 g/mL/peptide
Brefeldin A
1:10 in PBS
(1:50 - 1:100)
10 g/mL (1:50) or
Monensin
1:10 in PBS
(store at -20C)
Anti-CD3/ CD28
5
-
g/mL
(1:100)
brefeldinA
-
Note: 1 It is important to avoid solvent toxicity. Final DMSO+ethanol concentration from all sources (peptides, brefeldin
A, monensin) should not exceed 0.5%.
2
For most cytokines: use brefeldin A at 10 g/mL final concentration (see stock preparation table). For CD107 and
CD154: use monensin at 10 g/mL final concentration (see stock preparation table). For assays combining cytokines
and CD107 or CD154: use brefeldin A and monensin at 5 g/mL final concentration each.
3
CD28 and/or
CD49d (labeled antibody can be used if analysis of the marker is desired).
with
3.18.
3.19.
3.20.
Wash the cells with PBS at 1600 rpm, 8 min at room temperature.
Staining
3.21.
3.22.
3.23.
3.24.
3.25.
Discard supernatant and wash cells with PBS at 1600 rpm, 8 min,
room temperature.
3.26.
3.27.
3.28.
Prepare the surface staining cocktail according to titre per test for
each
Stain
Titre(ul/ sample)
X # of samples
Total ul
CD3
V500
10
50
CD4
CD8
3.29.
200
50
3.30.
3.31.
3.32.
3.33.
3.34.
3.35.
Add 2 ml per tube / well, mix well and incubate the cells for 10
min at room temperature, in the dark.
3.36.
3.37.
3.38.
3.39.
3.40.
3.41.
3.42.
3.43.
Antibody panel
Stain
Titre(ul/ sample)
X # of samples
Total ul
IFN-
IL-17
IL-4
TNF
IL-2
FITC
PE
APC
Alexa700
PE-Cy7
20
20
5
1.2
5
10
10
10
10
10
200
200
50
12
50
3.44.
3.45.
3.46.
3.47.
3.48.