Streptavidin pecy5 cy7

A total of 10⁵ WT and TNFR2 KO-MSCs were seeded in 96-well round-bottom plates (Falcon). Thereafter, they were immunostained using the following Abs: CD44-PE-Vio770, CD105-FITC, Sca1-APC, CD73-PE, CD90-Biotin or PE, anti-biotin-PE or VIOBLUE, CD45-VIOBLUE, CD34-Biotin or FITC (Miltenyi), and streptavidin-PeCY5/CY7 (eBioscience). We used isotypes Abs (Miltenyi) and unstained MSCs as controls for our experiments.
For F-actin staining, we have used F-actin Staining Kit, Green Fluorescence, Cytopainter (Abcam) according to supplier’s protocol. Briefly, we have seeded 10⁵ WT and TNFR2 KO MSCs in 96-well plates, treated them with 100 μl/well of Green Fluorescent Phalloidin Conjugate solution, and stained them in room temperature for 30 min. We then washed the cells with PBS 1× and analyzed in FITC channel using LSRFORTESSA flow cytometer (BD Biosciences). Data were then analyzed using FlowJo software v10 (FlowJo, LLC).

BM-MSCs were isolated from the femurs and tibias of 6- to 8-week-old C57BL/6 WT mice (Charles River and Envigo) and C57BL/6 TNFR2 KO (B6.129S2-Tnfrsf1b^tm1Mwm/J, The Jackson Laboratory). Mice were housed under pathogen-free conditions. Cells were cultured in 25-cm² flasks in MEMα medium (Gibco) containing low glucose, 1% GlutaMAX, 10% FBS, and 1% penicillin/streptomycin/neomycin (P/S/N) (Gibco). Cells were incubated at 37 °C in a 5% CO₂. Non-adherent cells were removed every 8 h; pure MSCs were obtained after 4–5 weeks. Cells were subcultured prior to confluency. In all experiments, WT and TNFR2 KO MSCs were used in passages 2 to 4.
For the identification of MSCs, 10⁵ cells/well WT and TNFR2-MSCs were seeded in Falcon 96-well round-bottom plates. They were then immunostained with CD44-PeCy7, Sca1-APC, CD105-FITC, CD73-PE, CD45-VIOBLUE, CD34-Biotin or FITC, CD90-Biotin or PE and anti-biotin-PE or VIOBLUE (Miltenyi), and streptavidin-PeCY5/CY7 (eBioscience). Unstained cells and isotypes were used as controls. Flow cytometric analysis was performed using the LSRFORTESSA flow cytometer (BD-Biosciences) and analyzed using the FlowJo software v10 (FlowJo-LLC).