Journal: Journal of Cellular and Molecular Medicine

Article Title: Extracellular calcium increases CXCR4 expression on bone marrow-derived cells and enhances pro-angiogenesis therapy

doi: 10.1111/j.1582-4934.2009.00691.x

Figure Lengend Snippet: Internalization of CXCR4 after binding with SDF-1. BMC were incubated with PBS with or without calcium for 4 hrs and then mixed with SDF-1 for 1 hr at 37° C. The surface (A) and intracellular (B) CXCR4 were measured by FACS. Surface CXCR4 was decreased and intracellular CXCR4 was increase after binding with SDF-1. * P < 0.05.

Article Snippet: BMCs (1 × 10 6 cells) were incubated with 0.5 mM CaCl 2 in PBS at 37°C for 4 hrs, then washed with PBS, re-suspended in 200 μl PBS containing recombinant mouse SDF-1α (final concentration 500 ng/ml, R&D Systems), and incubated at 37°C for 2 hr to allow SDF-1 to bind to the CXCR4 and the internalization of CXCR/DF-1 complex.

Techniques: Binding Assay, Incubation

Journal: Journal of Cellular and Molecular Medicine

Article Title: Extracellular calcium increases CXCR4 expression on bone marrow-derived cells and enhances pro-angiogenesis therapy

doi: 10.1111/j.1582-4934.2009.00691.x

Figure Lengend Snippet: BMC migration towards SDF-1. (A) BMCs were pre-incubated with PBS with or without CaCl 2 for 4 hrs at 37°C and transferred to the upper chamber of inserts in a 24-well plate containing DMEM and 100 ng/ml SDF-1. Migrated cells were counted after 6 hrs incubation at 37°C. Inhibitors AMD3100, LY294002, L-NMMA and anti-CXCR4 antibody were added to the upper chamber along with BMCs. * P < 0.05 versus Calcium treated group; # P < 0.05 versus other groups. (B) Effect of the inhibitors on CXCR4 surface expression. BMC were incubated at 37°C for 4 hrs in PBS without or with 0.5 mM CaCl 2 plus specified inhibitors, and subjected same FACS analysis as in Fig. . Amount of surface CXCR4 on BMC was expressed as MFI.

Article Snippet: BMCs (1 × 10 6 cells) were incubated with 0.5 mM CaCl 2 in PBS at 37°C for 4 hrs, then washed with PBS, re-suspended in 200 μl PBS containing recombinant mouse SDF-1α (final concentration 500 ng/ml, R&D Systems), and incubated at 37°C for 2 hr to allow SDF-1 to bind to the CXCR4 and the internalization of CXCR/DF-1 complex.

Techniques: Migration, Incubation, Expressing

Journal: Journal of Cellular and Molecular Medicine

Article Title: Extracellular calcium increases CXCR4 expression on bone marrow-derived cells and enhances pro-angiogenesis therapy

doi: 10.1111/j.1582-4934.2009.00691.x

Figure Lengend Snippet: Homing of injected BMC in ischemic site. BMCs from GFP mice were incubated in PBS with or without CaCl 2 at 37°C for 4 hrs, and then intravenously injected into WT BL6 mice with ischemic limb. SDF-1 protein (100 ng) was injected into the ischemic hindlimb muscle twice at consecutive days after the surgery. The hindlimb muscles were recovered 7 days after the cell injection, stained with DAPI for nucleus (blue), and examined for GFP cells incorporation under fluorescent microscopy. (A) Untreated BMC, (B) Ca-treated BMC, (C) BMC + SDF-1, (D) BMC/Ca + SDF-1. (E) The incorperated GFP cells were quantified as cells per high power field (HPF). * P < 0.05, and ** P < 0.01 versus all other groups ( n = 6).

Article Snippet: BMCs (1 × 10 6 cells) were incubated with 0.5 mM CaCl 2 in PBS at 37°C for 4 hrs, then washed with PBS, re-suspended in 200 μl PBS containing recombinant mouse SDF-1α (final concentration 500 ng/ml, R&D Systems), and incubated at 37°C for 2 hr to allow SDF-1 to bind to the CXCR4 and the internalization of CXCR/DF-1 complex.

Techniques: Injection, Incubation, Muscles, Staining, Microscopy

Journal: Journal of Cellular and Molecular Medicine

Article Title: Extracellular calcium increases CXCR4 expression on bone marrow-derived cells and enhances pro-angiogenesis therapy

doi: 10.1111/j.1582-4934.2009.00691.x

Figure Lengend Snippet: Effect of BMC injection on angiogenesis. BL6 mice with ischemia limb were injected intravenously with BM cells that were treated with or without CaCl 2 . SDF-1 protein was injected into the ischemic hindlimb muscle after the surgery. Blood flow of the lower limbs was measured using an laser Doppler perfusion images analyser. (A). Representative laser Doppler perfusion images at day 0 and day 21 after ischemia and cell injection. (B) Capillaries in the ischemic muscle from the mice at day 21 were identified by alkaline phosphatase cryosectional staining. (C). CD34 immunostaining of paraffin sections obtained from the ischemic muscle of mice at day 7. Brown spots are CD34 + cells (arrow). Blue represents cells counter-stained with haematoxylin. (D). Quantitative measurement of perfusion ratio of ischemic limbs to that of normal limbs at day 21 ( n = 6). (E) Quantification of capillary density on the tissue section, which is presented as the ratio of the number of capillaries to the number of muscle fibres. (F), Quantification of CD34 + cells around each muscle fibre. * P < 0.05 versus BMC/Ca group. # P < 0.05 versus other groups.

Article Snippet: BMCs (1 × 10 6 cells) were incubated with 0.5 mM CaCl 2 in PBS at 37°C for 4 hrs, then washed with PBS, re-suspended in 200 μl PBS containing recombinant mouse SDF-1α (final concentration 500 ng/ml, R&D Systems), and incubated at 37°C for 2 hr to allow SDF-1 to bind to the CXCR4 and the internalization of CXCR/DF-1 complex.

Techniques: Injection, Staining, Immunostaining