Cell migration of hVSMC and monocytes towards CXCL12 was assessed using the ChemoTx96-well cell migration system (30 µL well plate, 8 µm filter pore size; Neuroprobe, Gaithersburg, MD, USA). ChemoTX filters were pre-coated with 100 µg/mL Collagen IV for 48 h at 25 °C when hVSMC were tested. The bottom wells were loaded with 30 µL of test solutions (100 ng/mL CXCL12). Twenty-five microliters of cell suspension containing 105 cells were loaded onto the ChemoTX filter over each well and incubated for 2 h at 37 °C, 5% CO2. After incubation, transmigrated cells were washed, fixed and stained with Accustain Wright-Giemsa stain (Sigma-Aldrich). Stained cells were counted under a light microscope (400×) and average cell numbers were determined by counting the cells in three random non-overlapping vision fields. The chemotactic index was calculated as the ratio of cells that transmigrated through the filter in the presence versus absence (=PBS/control) of the test solutions.
Accustain wright giemsa stain
Manufactured by Merck Group
Accustain Wright-Giemsa stain is a laboratory reagent used in microscopy for the differential staining of blood cells. It is a combination of Wright's and Giemsa's staining methods, which together allow for the identification and differentiation of various types of blood cells, such as red blood cells, white blood cells, and platelets.
Automatically generated - may contain errors
3 protocols using accustain wright giemsa stain
1
Chemotaxis Assay for hVSMC and Monocytes
2
Automated Whole Blood Analysis
3
Erythroid Differentiation of Human iPSCs
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Human iPSCs were treated with 1 mg/mL dispase (Gibco) and replated onto the Martrigel-pretreated 12-well plate coated with mytomycin C-treated MEF and cultured in hESC medium at a density of 8 × 104 cells/well. The second day was defined as day 0. The medium was switched to STEMdiff APEL (STEMCELL Technologies) medium (AM). From day 0 to day 2, 50 ng/mL BMP4 and Activin A were added to the AM. At day 2, the cytokines in AM were changed to 50 ng/mL VEGF and 50 ng bFGF and kept for 2 d. From day 4 to day 6, 20 µM SB431542, and 50 ng/mL VEGF and bFGF were added to the AM (Wang et al. 2012 (link)). From day 6, the cells were cultured in AM supplemented with 50 μg/mL penicillin/streptomycin (Gibco), 50 ng/mL SCF, 50 ng/mL TPO, and 50 ng/mL IL-3, with a half-medium change every other day. For erythroid differentiation, the cells at day 13 were cultured in AM supplemented with 50 ng/mL hSCF, 20 ng TPO, 20 ng IL-3, and 5 units/mL EPO for 10 d, then collected and stained with Accustain Wright-Giemsa stain (Sigma-Aldrich). All the cytokines were purchased from Prospec.
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