F4 80 bv510

Differential cell counts were assessed by flow cytometry (BD LSRFortessa™ X-20 Cell Analyzer, BD Biosciences). BAL cells were added with Fixable Viability Stain 450 (562247, BD Biosciences) in a 96 V black microplate, incubated for 20 min at room temperature protected from light. After washing, Fc Block (553142, BD Biosciences) was added for another 20 min incubation time and after a new washing, membrane marker antibodies were finally added: CD11b-APC-Cy7 (557657), CD11c-PE-Cy7 (558079), F4/80-BV510 (743280) from BD Biosciences, Gr-1-PE-eFluor 610 (61-5931-82, eBioscience, Illkirch, France), CD45-Alexa (AF)700 (103128, BioLegend, Paris, France). Antibodies were incubated with BAL cells for 30 min at room temperature. After washing, microtubule-associated proteins 1A/1B light chain 3B (MAP1LC3B) and SQSTM1 expression were measured using the Autophagy detection reagent pack from Millipore (CF200097). Briefly, “reagent B” from Millipore was added to each well and cells were directly centrifuged 5 min at 300× g. Cells were washed with “assay buffer”, MAP1LC3B-FITC (from kit) and SQSTM1-AF647 (ab194721, Abcam, Cambridge, UK) were added to each well for 30 min protected from light. After a last wash, cells are resuspended in an “assay buffer” and flow cytometry was performed immediately.

For Cox8-mCherry-eGFP, Bio-Rad Ze5 Cell Analyzer was used. Cells were first sorted for mCherry positivity followed by eGFP. Analysis was done using FlowJo software. Flow cytometry analysis of immune cells was done using the Beckman Coulter MoFlo Astrios; immune cells from the colon were isolated by 25 mM EDTA digestion to remove epithelial cells, followed by a 0.5 mg/mL collagenase IV digestion, and were enriched for using a 40%–70% percoll gradient. Immune cells were stained for with CD45 APC eFluor 780, 1:200 (Invitrogen 47-0451-82); CD4 PECy7, 1:300 (eBioscience, 25-0041-82); CDllc FITC, 1:200 (BioLegend, 117305); CDllb APC, 1:250 (eBioscience, 17-0112-83); Ly6C V450, 1:300 (BD Biosciences, 560594), Ly6G PE, 1:300 (BD Biosciences, 560594), F4/80 BV510, 1:100 (BD Biosciences, 563633), 7AAD Percp Cy 5.5, 1:300 (BD Biosciences, 559925).