Apc conjugated mouse igg1 clone 11711

Cell surface expression levels of TRAIL receptors were analyzed by flow cytometry. Briefly, cells were detached from culture dishes by treatment with Accutase (Merck, Millipore, Darmstadt, Germany). Afterwards, cells were washed with cold MACS buffer (PBS supplemented with 2% fetal calf serum and 1 mM EDTA) and FcR-blocking was performed with human FcR blocking reagent (Miltenyi Biotec GmbH, Bergisch-Galdbach, Germany) according to the manufacturer’s instructions. For single stainings of TRAIL receptors, 2 x 10⁵ cells were incubated for 30 min at 4°C with the following APC-conjugated antibodies: anti-human TRAIL-R1 (clone #69036; 10 μg/ml), anti-human TRAIL-R2 (clone #71908; 10 μg/ml), anti-human TRAIL-R3 (clone #90906; 10 μg/ml) or anti-human TRAIL-R4 (clone #104918; 10 μg/ml), all purchased from R&D Systems GmbH, Wiesbaden, Germany. Respective isotype control stainings were performed with APC-conjugated mouse IgG₁ (clone #11711) and mouse IgG_2B (clone #13303) antibodies (both from R&D Systems GmbH). Finally, cells were washed twice in cold MACS buffer, resuspended in cold MACS buffer supplemented with 1% PFA and measured within 24 h using a FACSCalibur (Becton Dickinson, Heidelberg, Germany). A population size of 30,000 cells was regarded as representative for data evaluation using FlowJo v10 (FlowJo, LCC, Oregon, US).

Cell surface expression levels of TRAIL receptors were analyzed by flow cytometry. Briefly, the cells were detached from culture dishes by treatment with Accutase (Merck, Millipore, Darmstadt, Germany). Afterwards, the cells were washed with cold wash buffer (PBS supplemented with 0.5% BSA and 0.05% sodium azide) and FcR-blocking was performed with human FcR blocking reagent (Miltenyi Biotec GmbH, Bergisch-Galdbach, Germany), according to the manufacturer’s instructions. For single staining of TRAIL receptors, 2 × 10⁵ cells were incubated for 30 min at 4 °C with the following APC-conjugated antibodies: anti-human TRAIL-R1 (clone #69036; 10 µg/mL) or anti-human TRAIL-R2 (clone #71908; 10 µg/mL), which were both purchased from R&D Systems GmbH, Wiesbaden, Germany. Respective isotype control stainings were performed with APC-conjugated mouse IgG₁ (clone #11711) and mouse IgG_2B (clone #13303) antibodies (both from R&D Systems GmbH). Finally, the cells were washed twice in cold wash buffer, resuspended in cold wash buffer supplemented with 1% PFA, and measured within 24 h while using a FACSCalibur (Becton Dickinson, Heidelberg, Germany). A population size of 10,000 cells was regarded as representative for data evaluation using WEASEL v3.0.1 (WEHI, Melbourne, Australia).