Granulocyte-mediated T cell suppression

Granulocytes (Ly-6G⁺) were purified from the lungs of mice that were administered with B16F10 TCM or from the spleens of MC38 tumor-bearing mice using MACS Separation kit (Miltenyi Biotec). 1×10⁵ isolated granulocytes cells were incubated with OT-1 WT splenocytes by different ratio 1:1, 1:2, 1:4. The mixed cells were co-cultured in 96 well plate with SIINFEKL (0.5 ng/μl) peptide stimulation for 48 hr, and then ³H thymidine (PerkinElmer) was added (1 μl/well for 24 hr). The radioactivity of samples was counted with a TopCount NXT instrument (PerkinElmer) as previously described^{59 (link)}.

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Gui J., Zahedi F., Ortiz A., Cho C., Katlinski K.V., Alicea-Torres K., Li J., Todd L., Zhang H., Beiting D.P., Sander C., Kirkwood J.M., Snow B.E., Wakeham A.C., Mak T.W., Diehl J.A., Koumenis C., Ryeom S.W., Stanger B.Z., Puré E., Gabrilovich D.I, & Fuchs S.Y. (2020). Activation of p38α stress-activated protein kinase drives the formation of the pre-metastatic niche in the lungs. Nature cancer, 1(6), 603-619.

Publication 2020

MACS Separation kit 3H thymidine TopCount NXT instrument

Cells Granulocytes Lungs Mice Peptide Radioactivity Thymidine Tumor spleens

Corresponding Organization :

Other organizations : University of Pennsylvania, The Wistar Institute, University of Pittsburgh, University Health Network, Princess Margaret Cancer Centre, Case Western Reserve University, University School

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Variable analysis

independent variables

Granulocytes (Ly-6G+ cells) purified from the lungs of mice administered with B16F10 TCM or from the spleens of MC38 tumor-bearing mice
Ratio of isolated granulocytes to OT-1 WT splenocytes (1:1, 1:2, 1:4)

dependent variables

Proliferation of OT-1 WT splenocytes measured by 3H thymidine incorporation

control variables

SIINFEKL (0.5 ng/μl) peptide stimulation
Co-culture duration of 48 hours

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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