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