Apc mouse igg1

Flow cytometry was done as previously described [39 (link)]. Briefly, cells were washed with FACS-buffer (containing 0.01% sodium azide, 0.5% BSA, and 2 nM EDTA, all Sigma Aldrich) three times. For antibody incubation, cells were resuspended in 40 µL FACS-buffer supplemented with either 5 µL APC anti-human CD90 Antibody and 5 µL Alexa Fluor^® 488 anti-mouse/human CD44 antibody or their respective isotype controls (APC Mouse IgG1, κ Isotype Ctrl (FC) Antibody, Alexa Fluor^® 488 Rat IgG2b, κ Isotype Ctrl Antibody, all BioLegend, San Diego, CA, USA). After incubation on ice in the dark for 1 h, 1 mL FACS-buffer was added and the cells were centrifuged. The supernatant was discarded, and cells were resuspended in 500 µL FACS-buffer and measured using the FACS Canto II (BD Biosciences, Heildelberg, Germany). At least 50,000 events of each sample were recorded. Figures were created using FlowJo Version 10.7.1 (FlowJo LLC, Becton, Dickinson & Company, Franklin Lakes, NJ, USA).

Cells were cultured, left uninfected or DENV-infected, as described above. At 24 h post infection, cells were harvested, blocked in phosphate-buffered saline with 3% (w/v) bovine serum albumin and immunostained with control IgG (APC Mouse IgG1, cat 3400119, BioLegend) or anti-IFNAR1 (allophycocyanin (APC) anti-mouse IFNAR1, cat #127313, BioLegend)-conjugated antibody. Stained cells were fixed and subjected to flow cytometry (Accuri, BD systems, Franklin Lakes, NJ, USA). Staining was performed in triplicate and repeated on n=2 independent pMEF isolations.

When the isolated cells reached the third passage in the culture conditions, their cell surface antigen expressions were evaluated by flow cytometric analysis for characterization. For this purpose, MSC-specific markers CD44-PE and CD90-APC were analyzed as positive markers, and endothelial CD34-FITC and hematopoietic CD45-APC-Cy were used as negative markers for MSCs (BD the Biosciences, USA) [18 (link)]. Isotype antibodies determined for each antibody were used as negative control (Biolegend, San Diego; APC mouse IgG1, κ isotype control, APC/Cyanine7 mouse IgG1, κ isotype control, PE mouse IgG1, κ isotype control, FITC mouse IgG1, κ isotype control). Briefly, the cells were first collected by trypsinization and pelleted by centrifugation at 1500 rpm for 5 min. The number of viable cells was determined by staining the cells with trypan blue and counting. 10⁶ of cells were pipetted through a 70-μm filter, and the cells were separated from each other for flow cytometry analysis. After washing twice in staining buffer (2% FBS in PBS), primary and isotype antibodies were added to the mixtures and incubated in the dark for 30 min. After incubation, the cells were washed two more times with staining buffer and then analyzed by BD FACS Callibur [6 , 18 (link)]. This analysis was repeated three times, and the results are given as mean + SD.