Glioma Cell and Immune Cell Protocols

Murine glioma cells (GL261 and GL261-Luc) were a generous gift from Dr. G. Yancey Gillespie (UAB Brain Tumor Model Core Facility) and were grown in DMEM/F12 10% FBS, L-glutamine, and Pen/Strep as described [43 (link)]. Bone marrow derived macrophages (BMDM) were isolated and cultured as previously described [14 (link)]. For immunoblotting experiments, antibodies for phospho-STAT3 (Y705) and total STAT3 were purchased from Cell Signaling (9131; 79D7), and GAPDH from AbCam (ab8245). For IHC, rabbit anti-Iba1 was purchased from Wako (019-19741), rabbit anti-vWf from Millipore (AB7356) and rabbit anti-Ki67 from Abcam (AB15580). For flow cytometry experiments, antibodies for CD45 (30-F11), CD11b (M1/70), Gr-1 (RB6-8C5), F4/80 (BM8), CD4 (GK1.5), CD8 (53-6.7), Foxp3 (MF-14), CD25 (PC61) were purchased from BioLegend, iNOS (CXNFT) from eBioscience, and Arg1 (IC5868A) from R & D Systems [19 (link), 44 (link)]. The mouse cytokine/chemokine multiplex ELISA assay was purchased from EMD Millipore (MPXMCyto-70K).

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McFarland B.C., Marks M.P., Rowse A.L., Fehling S.C., Gerigk M., Qin H, & Benveniste E.N. (2016). Loss of SOCS3 in myeloid cells prolongs survival in a syngeneic model of glioma. Oncotarget, 7(15), 20621-20635.

Publication 2016

AB7356 AB15580 iNOS (CXNFT) MPXMCyto-70K

Antibodies Arg1 Assay Bone marrow derived macrophages Brain tumor Cd11b Cd25 Cells Chemokine Cytokine Elisa Flow cytometry Gapdh Glioma Inos L glutamine Mouse Rabbit Stat3 Strep

Corresponding Organization :

Other organizations : University of Alabama at Birmingham

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Variable analysis

independent variables

Murine glioma cells (GL261 and GL261-Luc)
Bone marrow derived macrophages (BMDM)

dependent variables

Phosphorylation of STAT3 (Y705)
Total STAT3 expression
Expression of Iba1, vWf, and Ki67
Levels of CD45, CD11b, Gr-1, F4/80, CD4, CD8, Foxp3, CD25, iNOS, and Arg1
Cytokine/chemokine levels

control variables

DMEM/F12 culture medium with 10% FBS, L-glutamine, and Pen/Strep
Experimental procedures for isolating and culturing BMDM

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

Annotations

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