Cd11b alexafluor488

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.

LPS (E. coli 0111:B4), dimethyl sulfoxide (DMSO), Tween-80, Hoechst 33342 and amino acid transporter inhibitor BCH (2-amino-2-norbornanecarboxylic acid) were bought from Sigma-Aldrich (St. Louis, MO, USA). Thioglycollate medium (Brewer modified) was obtained from Becton Dickinson (Sparks, MD, USA). Piperine was purchased from Guangzhou Institute for Drug Control (Guangzhou, China), dissolved in DMSO and stored at −20°C. Rabbit antibodies against phospho(p)-p70S6K, p70S6K, p-4E-BP1, 4E-BP1, p-S6(Ser235/236), GATA6, cleaved caspase-3, SLC7A5, SLC3A2, mTOR, and β-tubulin were purchased from Cell Signaling Technology (Danvers, MA, USA). The mouse antibody against LAMP2 was obtained from Abcam (Cambridge, MA, USA). PE-F4/80, FITC-CD11b, AlexaFluor488-CD11b, and APC-MHCII were obtained from eBioscience (San Diego, CA, USA). DMEM medium, fetal bovine serum (FBS), penicillin and streptomycin were products of Invitrogen (Carlsbad, CA, USA).
Female C57BL/6 mice were bought from the Experimental Animal Center of Southern Medical University (Guangzhou, China). Animal experiments were designed following National Institutes of Health guidelines and were approved by the Committee on the Ethics of Animal Experiments of Jinan University.

The experimental lungs and lung associated lymph nodes of knockout and wild-type animals were thoroughly minced and gently macerated in cold fresh medium. Organ fragments were re-suspended, and cold fresh medium was added. After a final re-suspension, samples were centrifuged and re-suspended in cold fresh medium. The cells were subjected to red blood cell lysis, washed in cold PBS, strained at 40 µm, and kept on ice until labeling. Flow cytometry analysis of lung inflammatory cells was made. Briefly, the immunostaining was performed by incubating the cells with the antibodies conjugated to fluorochromes: PerCP-Cy5.5-CD4 (Cat. 45-0042-82), Alexa Fluor 488-CD8 (Cat. 53-0081-82), Alexa-Fluor 488-CD11b (Cat. 53-0112-082), and EP-CD11c (Cat. 12-0114-82) (eBioscience, USA) at 1∶200 dilution for 60 minutes at 4°C. The cells were then washed twice in PBS, containing 1% bovine serum albumin, fixed, and analyzed by FACSCalibur flow cytometer (Becton Dicknson, USA). The region of living cells was determined using the parameters forward scatter versus side scatter. Ten thousand events were collected for each sample. Data were analyzed using WinMDI software (Scion Corporation, USA).