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

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.

1. Purchase MUTZ5 and/or MHH-CALL4 leukemia cell lines (TSLP responsive), recombinant human cytokine, and antibodies approved for use in phospho-flow cytometry assays to detect appropriate downstream phosphorylation targets.
2. Thaw harvested supernatant from control and cytokine-expressing stroma.
3. Plate the leukemia cell lines in R20 medium (see Table of Materials) at a concentration of 0.2 x 10⁶ cells per well in a 96-well tissue culture plate. Plate 3 wells per condition to allow for triplicate assays. Incubate for 2 h at 37 °C. This time allows for the loss of any phosphorylation that occurred during prior culture conditions.
   NOTE: 12 wells per cell line provides triplicate assays for each experimental condition shown in step 1.4.
4. After 2 h in culture, transfer the cells from each well to separate 4 mL tubes and centrifuge at 500 x g for 5 min, at 4 °C. Decant the supernatant.
   1. Resuspend the cells in 200 µL for each of the following experimental conditions (perform assays in triplicate): 1) negative control-media only, 2) positive control-media with recombinant cytokine at saturating concentration(s) (TSLP at 15 ng/mL, etc.), 3) control stroma-supernatant from control stroma, and 4) cytokine-stroma-supernatant from cytokine-producing stroma.
5. Incubate the cells for the duration specified by specific commercial phospho-assay (e.g. 30 min for phospho-STAT5 in MUTZ5 or MHH-CALL4 in response to TSLP).
6. Centrifuge at 500 x g for 5 min at 4 °C and decant the supernatant.
7. Perform phospho-flow cytometry staining per the manufacturer's protocol and collect flow cytometry data.
8. Analyze flow cytometry data.
   1. Gate on intact cells based on forward (FSC, indicates cell size) and side (SSC, indicates cell granularity) light scatter.
   2. Determine the median fluorescent intensity (MFI) of the intact cells in each sample. Compare the average MFI obtained from the triplicate values of stromal cell supernatant to that of positive and negative media controls.

Access restricted. Please log in or start a trial to view this content.

Materials

Name	Company	Catalog Number	Comments
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.