F actin staining kit

VIP (HSDAVFTDNYTRLRKQMAVKKYLNSILN, ≥98%) was synthesized by Sangon Biotech (Shanghai, China). Staurosporine (569397) and Ac-DEVD-CHO (235420) were purchased from Sigma-Aldrich. Full length human ZO-1 or caspase-3 was cloned into pcDNA3.1 (+). Sequence (5′ to 3′) of siZO-1: si-1: GUUAUACGAGCGAUCUCAU, si-2: GGAGGAAACAGCUAUAUGG. Sequence (5′ to 3′) of sicaspase-3: si-1: CCGACAAGCUUGAAUUUAU, si-2: GAAUUGAUGCGUGAUGUUU. The shRNA sequence that targets mouse ANXA1 sequence was designed as follows: 5′-GCCTCACAACCATCGTGAAGT-3′; adenovirus vectors expressing shANXA1 were constructed and generated by BrainVTA (Wuhan, China). The antibodies used in this study were as follows: anti–ZO-1 (Invitrogen, 33-9100), anti-cleaved caspase-3 (CST, 9661S), anti-caspase 3 (Proteintech, 19677-1-AP), anti-FLAG (CST, 14793S), anti-FLAG (Proteintech, 66008-3-Ig), anti-LAMP1 (CST, 15665S), anti-GAPDH (Proteintech, 60004-1-Ig), anti-β-actin (Proteintech, 60008-1-Ig), F-actin Staining Kit (Abcam, ab112127 and ab112125), and Fluorescein isothiocyanate-dextran (Sigma, FD70S).

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).