Ic0041g

The cells isolated from human GBM tumors were incubated with Alexa Fluor 488-conjugated mouse anti-human CD11b (R&D Systems, Minneapolis, MN; FAB16991G-100) and CD163-APC (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany; 130-100-612) or control immunoglobulin G (IgG) (Miltenyi, 130-098-846 and R&D Systems, IC0041G) for 30 min at 4 °C. FACS was performed to sort TAMs using a BD FACSAria II cell sorter (Becton, Dickinson and Company, NJ).

Adherent HCC-1954 cells (ShCTRL and ShTWIST) were processed into single-cell suspensions by trypsin digestion (0.25%, Sigma-Aldrich). Cell-surface staining was performed by incubating the cells with the following antibodies: AlexaFluor 488-conjugated anti-IL-17RC (FAB22691G, R&D Systems) and AlexaFluor 488-conjugated isotype control (IC0041G, R&D Systems), according to the manufacturer’s instructions. Intracellular staining was performed by a fixation step (PBS/Formaldehyde 0.05%) followed by permeabilization (PBS/Tween-20 0.05%) prior to intracellular staining with primary anti-Act1 (WW-18, SC-100647, Santa Cruz Biotechnology) or isotype control (SC-3878, Santa Cruz Biotechnology) antibodies and secondary antibody IgG2a-FITC (sc-358947, Santa Cruz Biotechnology), following the manufacturer’s instructions. Twenty thousand events were collected from each sample. The results were expressed as a percentage of positive cells that was calculated as experiment minus isotype control. All experiments were performed at least three times. Data were acquired using a FACSCalibur flow cytometer and analyzed using CellQuest software version 5.2 (BD Biosciences, San Jose, CA, USA).

Following siRNA transfection, HAECs were trypsinized, washed, and incubated in the presence of MMP14 Alexa Fluor® 488-conjugated antibody (0.1 µg per 10⁵ cells; #FAB9181G, R&D systems) or mouse IgG2B Alexa Fluor® 488-conjugated isotype control (0.1 µg per 10⁵ cells; #IC0041G R&D systems) for 40 minutes in fluorescence-activated cell sorting (FACS) buffer containing 2% FBS. Cells were then washed 2Xs with ice-cold FACS buffer, suspended 1% paraformaldehyde and analyzed by flow cytometry on a SH800S Cell Sorter and FlowJo software. The median fluorescence intensity less the isotype control was compared for each data set for two independent experiments.