Evaluation of Stromal Cytokine-Induced Intracellular Protein Phosphorylation Using Phospho-Flow Cytometry

Published: November 30, 2023

Abstract

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.

Protocol

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. Purchase MUTZ5 and/or MHH-CALL4 leukem…

Divulgations

The authors have nothing to disclose.

Materials

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.

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Citer Cet Article
Evaluation of Stromal Cytokine-Induced Intracellular Protein Phosphorylation Using Phospho-Flow Cytometry. J. Vis. Exp. (Pending Publication), e21808, doi: (2023).

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