Cd11b af488

Cells were incubated for 30 min on ice in 100 μl Hank’s balanced salt solution (Thermo Fisher) + 2% FBS with Fc block (1:100 dilution, clone 2.4G2; BD Biosciences) along with the following antibodies (all from BD Biosciences): CD45 APC-Cy7 (1:500, clone 30-F11), CD11b AF488 (1:500, clone M1/70), CD80 BV421 (1:200, clone 16-1OA1), and CD86 PE-Cy7 (1:500, clone GL1). IA/I-E AF647 (1:500, clone M5/114.15.2; BioLegend). Cells were then fixed and permeabilized using BD Cytofix/Cytoperm (BD Biosciences) according to manufacturer’s instructions. Cells were then incubated with an anti-RVFV antibody (kindly provided by Dr. Robert Tesh and the World Reference Center of Emerging Viruses and Arboviruses) at 1:500 dilution, followed by goat anti-mouse PE secondary antibody (1:1000, Santa Cruz biotechnology). Flow cytometry was performed using a FACSAria Fusion and data were analyzed using FlowJo software. Microglia, other myeloid lineage, and lymphocytes were resolved using CD45 and CD11b expression, with microglia identified as CD45^int CD11b^int, other myeloid as CD45^hi CD11b^hi, and lymphocytes as CD45^hi CD11b^– as previously described [65 (link)].

The mouse heart was perfused, excised, minced, digested with type I collagenase, mechanically disrupted, and filtered through a 70 μm cell strainer to get a single-cell suspension (21 (link)). Cells were collected by centrifugation, subjected to live-dead dye (LIVE/DEAD Fixable Yellow Dead Cell Stain Kit, Invitrogen, L34968) and surface antibody staining (fluorescent conjugated primary antibodies; BioLegend), and analyzed by flow cytometry. Cells were also sorted by flow cytometry to collect live CD45⁺ cells for transcriptomic studies. The following fluorescent antibodies were used in this study: CD45-PerCP (BD Biosciences, 561047), CD11b-AF488 (BD Biosciences, 557672), Ly6G-APC-Cy7 (BD Biosciences, 560600), F4/80-BV421 (BioLegend, 123137), and CD45-FITC (BioLegend, 103108).

Cultures of Isolated MSCs obtained from the different origins were labeled with
the following anti-human antibodies: CD11b-AF488, CD29-PE, CD73-PE, CD90-FITC,
CD105-PE (BD Bioscience), CD34-PE, CD19-PE, CD45-FITC (Beckman C), and HLADR-PE
from Invitrogen. Mouse isotype antibodies served as respective controls
(Invitrogen). Labelled cells were analyzed using a FACS-Vantage-SE flow
cytometry system running CellQuest software (BD). The fluorescence signals were
collected using logarithmic amplification.