human mesenchymal stem cell multi color flow cytometry kit  (R&D Systems)

Journal: The Journal of Physiology

Article Title: Endothelial‐to‐mesenchymal transition in the fetoplacental macrovasculature and microvasculature in pregnancies complicated by gestational diabetes

doi: 10.1113/jp287931

Figure Lengend Snippet: Figure 1. A-C, characterisation of primary HUVECs from non-GDM and GDM pregnancies using flow cytometry. A, representative flow cytometry plots to demonstrate the gating strategy applied to remove debris and doublets. Representative flow cytometry plot to demonstrate the gating of CD31+CD144+ HUVECs. B, percentage of total HUVECs from non-GDM and GDM pregnancies, that co-express CD31 and CD144 (n = 3 per group). Data are presented as the mean. C, histograms of fluorescence minus one (FMO) controls and stained HUVECs from non-GDM and GDM pregnancies expressing CD144 and CD31 (n = 3 per group). D-K, expression of EndMT markers in non-GDM and GDM HUVECs. HUVECs were isolated from non-GDM (n = 6) and GDM pregnancies (n = 5) and expression of EndMT markers, including endothelial and mesenchymal markers, were measured via RT-qPCR. As a mesenchymal cell positive control, primary pMSCs were used. Data are presented as the mean. Statistical analysis was performed using an unpaired t test (normally distributed), except TAGLN, which is presented as the median and statistical analysis was performed using a Mann–Whitney U test (not normally distributed) (∗P < 0.05 in GDM compared to non-GDM).

Article Snippet: Briefly, pMSCs were isolated using enzymatic digestion and were characterised using a human mesenchymal stem cell flow cytometry kit (R&D Systems, Minneapolis, MN, USA; catalog. no. FMC002) and tri-lineage differentiation (Kennedy, 2022). pMSCs were cultured in a humidified incubator at 37°C in 5% CO2, 20% O2 in low glucose Dulbecco’s modified Eagle’s medium (Gibco; catalog. no. 11885-092) supplemented with 10% fetal bovine serum (Gibco; catalog. no. 10270-106), 1% Antibiotic and Antimitotic (Gibco; catalog. no. 15240-062), and 1% Non-Essential Amino Acids (Gibco; catalog. no. 11140 035). pMSCs were passaged at 80–90% confluency using TrypLE Express (Gibco; catalog. no. 12563-029), which was inactivated with medium before seeding.

Techniques: Flow Cytometry, Fluorescence, Staining, Expressing, Isolation, Quantitative RT-PCR, Positive Control, MANN-WHITNEY