Pe conjugated f4 80

Mice (WT and Gsdmd^-/-) were intravenously challenged with 3 × 10⁵ CFU WT (GFP⁺) or candidalysin-deficient (ece1Δ/Δ) C. albicans. Three days after C. albicans infection, kidneys were homogenized using 40 µm cell strainers. Red blood cells were lysed with 1 ml ACK lysis buffer (Gibco) for 5 min at room temperature. Cells were then stained with PE-Cy7-conjugated CD45 (BioLegend), PE-conjugated F4/80 (BioLegend), and APC-conjugated CD11b (BioLegend) antibodies for 30 min. For cells isolated from WT (GFP⁺) C. albicans-infected mice, macrophages containing trapped C. albicans were recognized by GFP fluorescence. For cells isolated from candidalysin-deficient (ece1Δ/Δ) C. albicans-infected mice, the samples were fixed and permeabilized (BD Biosciences) after surface marker staining, and then stained with FITC-conjugated anti-C. albicans antibody (thermofisher) for 1 h. Flow cytometry was performed on the LSRFortessa (BD Biosciences) instrument. Flow cytometry data were analyzed with FlowJo software.

For the in vivo assessment of the effect of 3D-ADSC₁₀₀₀ and 2D-ADSC in attenuating inflammation in the FHF model, polarization of macrophage in the liver was evaluated using flow cytometry. After 7 h of cell transplantation, isolation of monocytes from the liver was conducted as described previously [31 (link)]. The isolated cells were stained with anti-mouse PE-Cy7-conjugated CD11b (Biolegend, San Diego, CA, USA), PE-conjugated F4/80 (Biolegend), APC-Cy7-conjugated CD86 (Biolegend), and PE-Cy7-conjugated CD206 (Biolegend) and analyzed by BD FACS Verse flow cytometer (BD Biosciences, San Jose, CA, USA). To check M1 and M2 phenotypes of macrophage, F4/80⁺ population was selected, and the expression of CD86 (M1 surface marker) and CD206 (M2 surface marker) was evaluated. Median fluorescence intensity (MFI) was calculated using FlowJo Software (FlowJo LLC, OR, USA). In addition, the concentration of IL-10 in serum was also evaluated by ELISA.

Aliquots of cells were transferred into a sterile 24-well plate with 1 mL of complete ISCOVES culture media supplemented with 2 mM L-glutamine, 5 mM Sodium Pyruvate, non-essential amino acids (MEM NEAA), 10 mM HEPES, 100 U/mL Penicillin, 100 U/mL Streptomycin and 2 × 10⁻⁵M of 2β-Mercaptoethanol. The cells were stimulated in vitro with 1 μL of cell stimulation cocktail (Tonbo Biosciences, San Diego, CA, USA). The plate was then placed into a HERAcell 150i incubator set at 37 °C and 5% CO₂ for 6 h. After incubation, the cells were harvested and spun at 300× g at 4 °C for 5 min.
The cells were washed with FACS buffer then incubated with Fc block in the dark at 4 °C for 20 min. Fluorescent antibodies were used against surface markers to label cells of interest: PE-Cy5-conjugated anti-CD11b (Tonbo 55-0112-U100) and PE-conjugated F4/80 (Biolegend, 123110) at 4 °C in the dark for 40 min. After incubation, cells were fixed with 300 μL of 2% paraformaldehyde. The cells were then permeabilized using permeabilization buffer (Tonbo Biosciences, San Diego, CA, USA) and stained with IFNγ (Biolegend, 505850). The cells were then resuspended with 200 μL of FACS buffer and filtered for analysis on the FC500 flow cytometer.