Flow Cytometric Analysis of Th1/Th17 Cells

For identification of IL17- and IFNγ-producing cells, lymphocytes were activated with 100 ng/ml phorbol myristate acetate (PMA) (Sigma-Aldrich), 1 µg/ml ionomycin, and 0.7 µl/ml Golgistop (BD Biosciences, San Jose, CA) in R-10P media (RPMI 1640 (Mediatech, Manassas, VA), 10% Fetal Calf Serum (Thermo Scientific, Rockford, IL), 1% penicillin/streptomycin, 0.1% β-mercaptoethanol, and 1% Glutamax (Fisher, Pittsburg, PA) for 5 hr at 37C. Staining was performed as previously described (Tanner et al. 2012 (link)). Briefly, the FcR were blocked via αCD16/32 (Biolegend, San Diego, CA) and CD4-APC was used as a cell-surface marker for Th1/Th17 lymphocytes. Permeabilization of cells allowed for the intracellular staining of IL-17-PE, IFNγ-FITC (Th17/Th1) and Foxp3-APC before fixation and FACS analysis on both Tregs and Teff cells (Biolegend). Cell-surface antibodies listed as CD4-FITC and CD25-PE were used as Treg markers.

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Wolf K.J., Daft J.G., Tanner S.M., Hartmann R., Khafipour E, & Lorenz R.G. (2014). Consumption of Acidic Water Alters the Gut Microbiome and Decreases the Risk of Diabetes in NOD Mice. Journal of Histochemistry and Cytochemistry, 62(4), 237-250.

Publication 2014

PMA Golgistop Fetal Calf Serum Glutamax αCD16/32

Cd25 Cell Fetal calf serum Fitc Ifnγ Il 17 Intracellular Ionomycin Lymphocytes Mercaptoethanol Penicillin Phorbol myristate acetate Streptomycin Surface antibodies Teff Th17

Corresponding Organization :

Other organizations : University of Manitoba, University of Alabama at Birmingham

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Variable analysis

independent variables

Phorbol myristate acetate (PMA) concentration (100 ng/ml)
Ionomycin concentration (1 µg/ml)
GolgiStop concentration (0.7 µl/ml)

dependent variables

Identification of IL17- and IFNγ-producing cells
Th1/Th17 lymphocyte population
Treg and Teff cell populations

control variables

R-10P media composition (RPMI 1640, 10% Fetal Calf Serum, 1% penicillin/streptomycin, 0.1% β-mercaptoethanol, 1% Glutamax)
Incubation time (5 hours)
Incubation temperature (37°C)

controls

FcR blocking via αCD16/32
Positive control: CD4-APC as a cell-surface marker for Th1/Th17 lymphocytes
Positive control: Foxp3-APC for Tregs
Positive control: CD4-FITC and CD25-PE for Treg markers

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