Figure 1.

Enhanced CXCR4 receptor expression on NaHS treated BMMNCs and chemotaxis of HSPCs toward SDF-1α. (a) Representative experimental setup for evaluating CXCR4 expression and other parameters. Total BMMNCs were isolated and treated with either vehicle or NaHS for 20 min, washed and cultured in IMDM+10% HI-FBS for 24 h. (b) mRNA amplification plots of CXCR4 receptor on BMMNCs. Relative mRNA expression was calculated using Livak method and β-Actin as a house keeping gene. Time course plot for CXCR4 expression after 12, 24 and 36 h of treatment with vehicle or NaHS to optimize the time point for maximum CXCR4 expression after treatment. (c) Changes in the CXCR4 expression in vehicle/NaHS (100 and 300 μM) treated BMMNCs after 24 h using real time PCR. Data are expressed as mean ± SEM fold change in expression with respect to control (n = 4) and the comparisons were done using one-way ANOVA. Lin^neg cells were sorted from freshly isolated BMMNCs, exposed to NaHS/vehicle, washed and cultured in media for 24 h (37ºC, 5%CO₂, 95% humidity). Thereafter, the cells were washed and resuspended in IMDM+0.5%BSA and were allowed to migrate for 4 h. The number of different subsets of HSPCs migrated to the bottom chamber in the presence or absence of SDF-1α were quantified using flow cytometry; (d)Total Lin^neg cells migrated (e), Migration of gated LSK population (f), Migration of LT-HSCs (CD34⁻) and (g) total ST-HSCs (CD34⁺) migrated. Data are expressed as mean ± SEM total number of cells migrated. (n = 3, ns = non-significant *p ≤ 0.05, **p ≤ 0.01 and ***p ≤ 0.001) and analyzed using one-way ANOVA