Facscan cytometer canto 2

The cells were blocked with anti-CD16/32 for 20 min to reduce non-specific binding. For the analysis of EPOR, CD80, CD86, and CD206 levels at the cell surface, harvested cells were incubated with anti-EPOR, anti-CD86, anti-CD80, anti-CD206 antibody, and anti-F4/80 antibody on ice for 30 min, washed twice, and then taken to flow cytometry. For the analysis of intracellular TNF-α, IL-6, and IL-10 levels, harvested cells in BALF from eCIRP-treated mice were incubated with anti-F4/80 antibody for 30 min. Subsequently, the cells were permeabilized and fixed with Cytofix/Cytoperm™ Fixation/Permeabilization kit (BD, #554714) and further stained with anti-TNF-α, anti-IL-6, and anti-IL-10 antibody on ice for 30 min, washed twice, and then taken to flow cytometry. Flow cytometry was performed using a FACScan cytometer CANTO II (Becton Dickinson, Franklin Lakes, NJ, USA) or Gallios (Beckman Coulter, USA). Data were collected using CellQuest software (Becton Dickinson) or Kaluza software (Beckman coulter) and analyzed with FlowJo software (Tree Star, Inc.). Macrophages in BALF were marked by F4/80, and the MFIs and percentage of target proteins were measured.

BMDMs were incubated with the PE anti-CD91 antibody (1:100) for 30 min on ice, washed twice, and subjected to flow cytometry. For the in vivo evaluation, cells derived from alveolar lavage fluid of ARDS mice were incubated with PE anti-CD91 antibody (1:100), APC/Cyanine7 F4/80 antibody (1:100), PerCP CD11b antibody (1:100), and APC CD11c antibody (1:100) for 30 min on ice, washed twice, and subjected to flow cytometry. The mean fluorescence intensity of CD91 was assessed in F4/80⁺CD11c⁺CD11b⁻ cells. Flow cytometry analysis was performed using FACSCanto™ II Clinical Flow Cytometry System (BD Biosciences). The flow cytometric data were collected using a FACScan cytometer CANTO II (Becton Dickinson, Franklin Lakes, NJ, USA) and analyzed using FlowJo software (TreeStar, Ashland, OR, USA).

The apoptotic thymocytes were harvested after the thymocytes were treated with 1 mM dexamethasone (MCE, #HY-14648) at 37°C for 6 h. The macrophage-mediated efferocytosis in vitro was evaluated using immunofluorescence staining and flow cytometry. For immunofluorescence staining, the Rab43-C or Rab43-cKO macrophages were seeded in cell culture dishes (NEST Biotechnology, Wuxi, China; #801002) overnight. The macrophages were incubated with apoptotic thymocytes labeled with carboxyfluorescein succinimidyl ester (CFSE; 10mM, 1:1,000) at a 1:10 ratio and then cultured in PBS at 37°C for 120 min. The macrophages were subsequently stained with Dil Stain (1:1,000). The cells were viewed using a Leica confocal microscope (Leica Microsystems).
For flow cytometry, the BMDMs were incubated with CFSE-labeled apoptotic thymocytes at a 1:10 ratio and then cultured in PBS at 37°C for 120 min. The macrophages were labeled with the anti-F4/80 antibody (1:200). The flow cytometric data were collected using a FACScan cytometer CANTO II (Becton Dickinson, Franklin Lakes, NJ, USA) and analyzed using FlowJo software for Windows (Tree Star).