Monoclonal antibodies against cd

Cells from freshly collected cloudy peritoneal effluents were acquired on an eight-colour FACSCanto II (BD Biosciences) and analyzed with FlowJo 10.1 (TreeStar), using monoclonal antibodies against CD3 (SK7) and CD15 (HI98 or HIM1) from BD Biosciences; and anti-CD14 (61D3) from eBioscience. Leukocyte populations were gated based on their appearance in side scatter and forward scatter area/height and exclusion of live/dead staining (fixable Aqua; Invitrogen).

Adipose derived mesenchymal stem cells were isolated and cultured from the groin adipose tissue of adult C57BL/6 mice (body weight: 28.20 ± 0.66 g), which were obtained from the Experimental Animal Center of Xinxiang Medical University. The upon experimental methods and reagents used were referred to the previously published protocol (Zuk et al., 2001 (link); Li et al., 2013 (link)). Cells were cultured with low sugar DMEM (Dulbecco’s modified Eagle’s medium) containing 10% FBS (Fetal bovine serum; Hyclone, United States). When cells reached 80% confluence, they were passaged using 1:2 splits. The morphology of the cells was observed by inverted microscope. Immunofluorescence and flow cytometry analysis were conducted on passage 3 AD-MSCs using monoclonal antibodies against CD29, CD44, CD73, and CD45 (BD Bioscience, 1:500).
CD73⁺AD-MSCs and CD73^– AD-MSCs were isolated using flow cytometry and then cultured. With CD73 as the standard, flow cytometry (FACS Aria II, BD Bioscience) was used to sort out the two subgroups. The morphology of CD73⁺ AD-MSCs and CD73^–AD-MSCs in primary culture was determined by using HE staining. Subpopulations were further subcultured and the cells from the fourth to sixth passages were used for the follow-up experiments. Quantitative real-time PCR and Western blotting were used to detect the expression of CD73 in the two subgroups.