A | B | C | |
Reagent | Final concentration | Amount | |
PBS | n/a | 100mL/well | |
Anti-CD3e (0.5mg/mL) | 2.5mg/mL | 0.5mL/well | |
Anti-CD28 (1mg/mL) | 1mg/mL | 0.1mL/well |
A | B | C | |
FACS buffer | |||
Reagent | Final concentration | Amount | |
PBS | n/a | 490mL | |
FBS | 2% | 10mL | |
Total | n/a | 500mL |
A | B | C | |
T cell culture media | |||
Reagent | Final concentration | Final concentration | |
MEM⍺ | n/a | 429mL | |
FBS | 10% | 50mL | |
GlutaMAX 100× | 2× | 10mL | |
HEPES, 1M | 0.02M | 10mL | |
2-mercaptoethanol (55 mM) | 55μM | 0.5mL | |
Normocin, 1g (50mg/ml) | 50μg /ml | 0.5mL | |
Total | n/a | 500mL |
A | B | C | |
Th0 differentiation cocktail (2.5×) | |||
Reagent | Final concentration | Amount | |
T cell culture media | n/a | 1mL | |
IL-2 (5×105U/mL) | 100U/mL | 0.5mL |
A | B | C | |
Th1 differentiation cocktail (2.5×) | |||
Reagent | Final concentration | Amount | |
T cell culture media | n/a | 1mL | |
IL-2 (5×105U/mL) | 100U/mL | 0.5mL | |
IL-12 (10μg/mL) | 4ng/mL | 1mL | |
Anti-IL-4 (10mg/mL) | 10μg/mL | 2.5mL |
A | B | C | |
Th2 differentiation cocktail (2.5×) | |||
Reagent | Final concentration | Amount | |
T cell culture media | n/a | 1mL | |
IL-2 (5×105U/mL) | 100U/mL | 0.5mL | |
IL-4 (10μg/mL) | 10ng/mL | 2.5mL | |
Anti-IFNg (10mg/mL) | 10μg/mL | 2.5mL |
A | B | C | |
Th9 differentiation cocktail (2.5×) | |||
Reagent | Final concentration | Amount | |
T cell culture media | n/a | 1mL | |
IL-2 (5×105U/mL) | 100U/mL | 0.5mL | |
IL-4 (10μg/mL) | 10ng/mL | 2.5mL | |
Anti-IFNg (10mg/mL) | 10μg/mL | 2.5mL | |
TGF-b (5μg/mL) | 1ng/mL | 0.5mL | |
IL-1b (10μg/mL) | 10ng/mL | 2.5mL |
A | B | C | |
Treg differentiation cocktail (2.5×) | |||
Reagent | Final concentration | Amount | |
T cell culture media | n/a | 1mL | |
IL-2 (5×105U/mL) | 300U/mL | 1.5mL | |
Anti-IFNg (10mg/mL) | 10μg/mL | 2.5mL | |
TGF-b (5μg/mL) | 5ng/mL | 2.5mL | |
Anti-IL-4 (10mg/mL) | 10μg/mL | 2.5mL |
A | B | C | |
MEM⍺ | Fisher Scientific | Cat#32-571-101 | |
FBS | R&D Systems | Cat#S11150 | |
GlutaMAX 100× | Fisher Scientific | Cat#35050061 | |
HEPES | Fisher Scientific | Cat#MT25060CI | |
2-mercaptoethanol | Fisher Scientific | Cat#21985023 | |
Anti-CD3e | Cytek Biosciences | Cat#145-2C11 | |
Anti-CD28 | BD Biosciences | Cat#553294 | |
IL-2 | Prometheus Laboratories | Cat#NDC65483-116-07 | |
IL-12 | PeproTech | Cat#210-12 | |
TGF-b1 | BioLegend | Cat#763104 | |
IL-1b | R&D Systems | Cat#401-ML/CF | |
IL-4 | PeproTech | Cat#214-14 | |
Anti-IL-4 | BioXCell | Cat#BE0045 | |
Anti-IFNg | BioXCell | Cat#BE0055 | |
Mouse CD4+ T-cell Isolation Kit | STEMCELL Technologies | Cat#19852 | |
Mouse IFN-gamma DuoSet ELISA | R&D Systems | Cat#DY485 | |
Mouse IL-4 DuoSet ELISA | R&D Systems | Cat#DY404 | |
Mouse IL-9 DuoSet ELISA | R&D Systems | Cat#DY409 | |
Foxp3/transcription factor staining buffer set | Fisher Scientific | Cat#00-5523-00 | |
Anti-CD4-eFluor 450 | eBioscience | Cat#48–0042–82 | |
Anti-CD25-PerCP | TONBO Biosciences | Cat#65-0251 | |
Anti-Foxp3-PE | Fisher Scientific | Cat#12-5773-82 | |
PMA | Sigma-Aldrich | Cat#P1585 |
A | B | C | |
Naïve CD4+ T cells | 5×104/well | 100μL/well | |
T cell medium | 50μL/well | ||
Total | Total | 150μL/well |
A | B | C | |
Final concentration | Amount | ||
T cells | 1×106/mL | 100μL | |
T cell Medium | n.a | 100μL | |
PMA (50μg/mL) | 50ng/mL | 0.2μL |
A | B | |
FACS buffer | 50µL | |
Anti-CD4-eFluor 450 | 0.3µL | |
Anti-CD25-PerCP | 0.3µL |
A | B | |
1×Permeabilization Buffer | 50µL | |
Anti-Foxp3-PE | 0.3µL |