Ly6c apc

Anti-APC Multisort Kit (Milteyi Biotec; #130-091-255), Ly6C-APC (eBiosciences; clone HK1.4), CD115 Microbead Kit (Milteyi Biotec; #130-096-354), and LD Columns (Milteyi Biotec; #130-042-901) were used. Murine bone marrow cells were harvested and cultured for 2 days as previous described. 3x10⁷ cells were recovered and sorted according to manufacturer’s instructions. Cells were treated with FC blocking buffer and stained with Ly6C-APC and CD115-biotin. After incubation and washing, cells were incubated with anti-APC beads and passed through an LD column. The flow through and retained factions were collected, and anti-APC beads were cleaved with Release Buffer. Both factions were then incubated with Stop Buffer and anti-Biotin beads. The two factions were passed through a second LD column, resulting in 4 fractions based on Ly6C and CD115 profiles.

For FACS analysis, all the cells were stained in the presence of an Fc block. For staining, cells were incubated for 20 min at 4 °C using the following antibodies: CD11b-FITC, Ly6C-APC, Ly6G-APC, F4/80- PE, CD11c-PE, CD3-APC, CD8-FITC, CD4-FITC, CD19-FITC (all from eBiosciences, CA, USA) and CD8-PerCP-Cy5.5, CD4-PerCP-Cy5.5 (all from BD Biosciences). The following markers were used to identify specific cell types: monocytes (CD11b+ Ly6C+), neutrophils (CD11b+ Ly6G+), macrophages (F4/80), dendritic cells (CD11c), B cells (CD19), CD4+ T cells (CD3+ CD4+) and CD8+ T cells (CD3+ CD8+).
Unstained cells were used to set up the instrument. Compensation controls were set up using single stains and isotype controls were used to determine the level of non-specific binding. The cells were gated based on simple forward and side scatter patterns. Furthermore, all the dead cells were excluded using the viability dye, propidium iodide and doublet discrimination was performed by plotting FSC-H vs. FSC-A. If we take the example of lymphocytes, they were identified and gated by their forward and side scatter patterns. The CD3+ T cells were then further identified and gated by the expression of CD4+ and CD8+. The expression of surface markers was assessed using CellQuest analysis software on a FACSCalibure flow cytometer (BD Biosciences).

Cisplatin, doxorubicin and VX‐765 were purchased from Selleck. Ac‐DEVD‐CHO was obtained from MedChem Express. Propidium iodide (PI), Hoechst 33342, dimethyl sulfoxide (DMSO) and Tween‐20 were bought from Sigma‐Aldrich. Lipofectamine RNAiMAX, Dulbecco's Modified Eagle's Medium (DMEM) medium with high glucose, Opti‐MEM, foetal bovine serum (FBS), streptomycin and penicillin were the products of ThermoFisher. The antibody against actin was purchased from Santa Cruz. The antibodies against cleaved caspase‐3, caspase‐3, cleaved caspase‐7, cleaved caspase‐8, cleaved caspase‐9, PARP and horse‐radish peroxidase (HRP)‐linked IgG were purchased from Cell Signaling Technology. Antibodies against pro‐caspase1+p10+p12, GSDMD (clone: EPR19828) and DFNA5/GSDME were obtained from Abcam. F4/80‐PE, Ly‐6C‐APC and CD11b‐AlexaFluor488 were obtained from eBioscience. F4/80‐AlexaFluor647 was bought from BioLegend.

Single-cell suspensions of BM and skin-infiltrating leukocytes were incubated at 4 °C for 20 min in staining buffer (1 × PBS with 0.1% bovine serum albumin and 0.1% sodium azide) containing the appropriate antibody mix. NOS2-AF488, CD45.1-eFluor 450, Ly6G-FITC, Ly6G-PE, Ly6G-PE.Cy7, CD11b-PE, CD11b-APC, F4/80-PE, CD115-APC, Ly6C-APC, CD45-PE, B220-PE, CD4-APC, CD8-APC, and CD105-APC antibodies were obtained from eBioscience. Lineage cocktail-APC, CD11c-APC.Cy7, α-BrdU-FITC and CD34-PE antibodies were purchased from BD Pharmingen (San Diego, CA, USA). Arginase-FITC antibody was purchased from R&D systems (Minneapolis, MN, USA). For the flow cytometric analysis of Lineage-positive cells, the cells were stained with biotinylated Lineage cocktail obtained from BD Pharmingen, then labeled with streptavidin-V450 (BD Pharmingen). Data were collected using a FACSCalibur or LSRII-Green (both BD Pharmingen) and were analyzed using the FlowJo software (Tree Star, Ashland, OR, USA).