Source: Coats, J. S., et al. Expression of Exogenous Cytokine in Patient-derived Xenografts via Injection with a Cytokine-transduced Stromal Cell Line. J. Vis. Exp. (123) (2017).
This video demonstrates the evaluation of stromal cytokine-induced intracellular phosphorylation of proteins in leukemia cells. These intracellular phosphorylated proteins are quantified using the phospho-flow cytometry technique.
1. Phospho-flow Cytometry Assays to Evaluate the Activity of Cytokine Produced by Stroma
NOTE: Assess the supernatant from the engineered stroma before the first experiment to verify the relevant bio-activity of the stroma-generated cytokine for the individual experiment. Once the engineered stroma is validated, further testing is not necessary unless stroma are introduced that were produced with a new vector.
The authors have nothing to disclose.
10 mL serological pipettes | Falcon | 357551 | |
15 mL polypropylene conical tubes | Fisher | 05-539-12 | |
50 mL polypropylene conical tubes | Falcon | 352098 | |
1 mL pipette tips | Fisher | 2707509 | |
200 μL pipette tips | Denville Scientific | P3020-CPS | |
10 μL pipette tips | Denville Scientific | P-1096-CP | |
“R20” cell culture medium, % of total volume (makes 195 mL) | Lab Recipe | ||
RPMI, 76.84% (150 mL) | Mediatech | 10-040-CV | |
FBS, 20.49% (40 mL) | Mediatech | 35-011-CV | |
2mM L-glutamine, 1.02% (2 mL) | Mediatech | 35-011-CV | |
1 mM Na pyruvate, 1.02% (2 mL ) | |||
Penicillin-Streptomycin, 0.51% (1 mL | Mediatech | 30-002-Cl | |
2-Mercaptoethanol, 0.10% (0.1 M), 0.10% (200 µL) | MP | 190242 | |
Biologics | |||
"CALL-4" MHH-CALL-4 cell line, human B cell precursor leukemia | DSMZ | ACC 337 | |
MUTZ-5 cell line, human B cell precursor leukemia | DSMZ | ACC 490 | |
Recombinant Human TSLP protein | R&D Systems | 1398-TS-010 | |
Flow cytomery antibodies (clone)* | |||
Anti-mouse CD45 FITC (30F11) | Miltenyi Biotec | 130-102-778 | |
CD127 PE (MB15-18C9) (alternate name is IL-7Rα) | Miltenyi Biotec | 130-098-094 | |
CD34 APC (8G12) | BD Biosciences | 340667 | |
CD45 PE-Cy7 (HI30) | eBioscience | 25-0459 | |
"FVD" Fixable viability dye eFluor 780 | eBioscience | 65-0865 | |
Ig κ light chain FITC (G20-193) | BD Pharmingen | 555791 | |
Ig λ light chain FITC (JDC-12) | BD Pharmingen | 561379 | |
IgD PE (IA6-2) | BioLegend | 348203 | |
IgM PE-Cy5 (G20-127) | BD Biosciences | 551079 | |
pSTAT5 PE, mouse anti-STAT5 (pY694) | BD PhosphoFlow | 612567 | |
Other materials & equipment | |||
FlowJo flow cytometry analysis software | FlowJo, LLC | Version 10 | |
*All antibodies are anti-human unless otherwise stated. |