Cells were plated onto 6-well plates at a density of 2 × 104 cells/well. The following day, cells were washed and incubated in the presence and absence of various test compounds. At selected times, cells were dissociated from the plate with trypsin and counted in a Beckman Z1 Coulter Counter (Beckman Coulter Incorporated, Brea, CA). Cell proliferation was also monitored by measuring the incorporation of radiolabeled thymidine into DNA [26 (link)]. For these experiments, cells were pulsed with [3H]thymidine for 4 hours, washed with PBS, and fixed with trichloracetic acid (10%). DNA was then solubilized with 0.2% SDS/0.2N NaOH and radioactivity quantified by scintillation spectroscopy [26 (link)].
Beckman z1 coulter counter
Manufactured by Beckman Coulter
Sourced in United States
The Beckman Z1 Coulter Counter is a laboratory instrument used for automated cell counting and sizing. It operates by drawing a sample through a small aperture and detecting changes in electrical impedance as cells or particles pass through, providing accurate and reproducible cell count and size data.
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4 protocols using beckman z1 coulter counter
1
Cell Proliferation Assay Protocol
2
Neonatal Rat Cardiac Fibroblast Proliferation
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Cardiac fibroblasts from neonatal rat were seeded (2 × 106 cells/well) onto 6-well plates in serum-containing media. After 24 hr, cells were washed and treated with serum-containing media in the presence and absence of various test compounds.
Cell number determinations were performed at various times by dissociating cells with trypsin and counting cells in a Beckman Z1 Coulter Counter (Beckman Coulter). For detection of cardiac fibroblast proliferation by measuring DNA synthesis, cells were incubated with [3H]thymidine for 4 hr, washed three times with ice-cold PBS, and fixed with 10% trichloracetic acid at 4°C for 30 min. DNA was then extracted with 0.2% SDS/0.2 N NaOH and radioactivity was determined by scintillation spectroscopy.
Cell number determinations were performed at various times by dissociating cells with trypsin and counting cells in a Beckman Z1 Coulter Counter (Beckman Coulter). For detection of cardiac fibroblast proliferation by measuring DNA synthesis, cells were incubated with [3H]thymidine for 4 hr, washed three times with ice-cold PBS, and fixed with 10% trichloracetic acid at 4°C for 30 min. DNA was then extracted with 0.2% SDS/0.2 N NaOH and radioactivity was determined by scintillation spectroscopy.
3
Evaluating Endothelial Cell Proliferation
4
Cell Viability Assay in Cardiomyocytes
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The RealTime-Glo MT Cell Viability Assay (Promega, Madison, WI, USA, G9711) was performed in cultured neonatal cardiomyocytes, using three NC-RNA and three shRNA cell samples, to evaluate possible differences between the two conditions. Cells were collected through a 5 min incubation procedure with Trypsin-EDTA and then counted using the Beckman Z1 coulter counter (Beckman Coulter, Brea, CA, USA). A final number of 2000 cells of each condition were incubated for 30 min at 37 °C with 20 µL of RealTime-Glo reagent in DMEM medium. Luminescence was measured using a Modulus Luminometer (Promega, Madison, WI, USA). For this study, the cell viability assay was first measured in cultured cardiomyocytes that were not infected with lentiviral particles as a control to confirm that the transduction did not affect viability.
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