Alyses (caspase-1 activity and loss of membrane integrity), add two g/mL PI for the cells

Alyses (caspase-1 activity and loss of membrane integrity), add two g/mL PI for the cells from the second nicely treated with FLICATM and analyze promptly as such assays can not be performed applying fixed cells. For a single-color compensation manage (loss of membrane integrity), use the cells from the third well (not treated with FLICATM), add two g/mL PI and analyze promptly. Measure your cells in a suitable flow cytometer, such as the BD FACSCaliburTM. Components BxPC-3 pancreatic adenocarcinoma cell line: ATCCCRL-1687TM, American Variety Culture Collection, Manassas, VA, USA. Twelve-well plates: CELLSTARCell Culture Multiwell Plates, catalog no. 665180, Greiner Bio-One, Frickenhausen, Germany. RPMI 1640 medium: catalog no. 52400, ThermoFisher Scientific, Dreieich, Germany. FBS: catalog no. 10270, ThermoFisher Scientific, Dreieich, Germany. L-Glutamine: catalog no. K 0202, Merck, Berlin, Germany. Sodium pyruvate: catalog no. 11360, ThermoFisher Scientific, Dreieich, Germany. Penicillin/streptomycin: catalog no. A 2212, Merck, Berlin, Germany. Pyroptosis/Caspase-1 Assay Kit containing nigericin, FAM-YVAD-fmk, Cellular Wash Buffer and Fixative, catalog no. 9146, ImmunoChemistry Technologies, Bloomington, MN, USA.Author Manuscript Author Manuscript Author Manuscript Author Manuscript14.15. 16.17. 18.19.20. 7.four.four Eur J Immunol. Author manuscript; readily available in PMC 2020 July 10.Cossarizza et al.PageFalcon12 75mm, five mL polystyrene round bottom test tubes: catalog no. 352054, Corning, Wiesbaden, Germany. StemProTM AccutaseTM Cell Dissociation Reagent: catalog no. A1110501, ThermoFisher Scientific, Dreieich, Germany. PI: catalog no. P4170, Merck, Darmstadt, Germany. BD FACSCaliburTM Flow Cytometer: BD MMP-9 Activator Purity & Documentation Biosciences, Heidelberg, Germany. BD CellQuestTM Pro PPARα Activator Purity & Documentation computer software: BD Biosciences, Heidelberg, Germany.Author Manuscript Author Manuscript Author Manuscript Author Manuscript7.four.five Data evaluation: Information is often acquired employing the acquisition application supplied with the flow cytometer, for example, the BD CellQuestTM Pro computer software. Evaluation is usually accomplished with either the computer software applied for data acquisition or with any appropriate FCM information analysis software program. For information analysis, adhere to the manual offered using the Pyroptosis/Caspase-1 Assay Kit. The FAM-FLICA reagent is excited at 488 nm (blue laser) and includes a peak emission at 51535 nm. Therefore, FAM-FLICA fluorescence is often detected applying a BP filter 530/30 (FL1 channel with the FACSCalibur flow cytometer). For single-staining, instrument settings need to be set for the FAM-FLICA fluorescence channel on logarithmic fluorescence scale as well as the threshold need to be set on FSC to exclude compact cellular fragments and debris. For sample acquisition and analysis, two sequential plots are necessary: an FSC-H versus SSC-H dot plot to gate on the population of interest (gate A) along with a histogram plot with the log FAM-FLICA fluorescence on the x-axis versus the amount of cells around the y-axis set on gate A. Adjust the voltage from the FAM-FLICA fluorescence channel, if essential, to ensure that fluorescence is on scale and caspase-negative (FAMFLICA-) cells seem within the lower log fluorescence output decades in the x-axis. Caspase-positive (FAM-FLICA+) cells will seem as a shoulder or as a separate peak shifted to the proper side on the unfavorable peak. A typical outcome demonstrating a histogram overlay of adverse and constructive cells is shown in Fig. 42A. The gate is set on FAM-FLICA-positive cells and their percentages in untreated and nigeric.