For proliferation assays, 50,000 cells per well were seeded in a 6-well plate, and cell number was counted using a Cellometer Vision cytometer (Nexcelom Bioscience) on the days indicated by the trypan blue exclusion method. To measure cell viability using CellTiter-Glo (Promega), 5000 cells/well were seeded in 96-well plates. For focus formation assays, 1000 RMS cells were seeded in 10 cm plates in the presence of 1 million NIH 3T3 cells, and medium containing IPTG or doxycycline was replenished every 72 h. Foci were stained 2 weeks later with Giemsa solution (Sigma) and counted using ImageJ software (National Institutes of Health [NIH]).
Cellometer vision cytometer
Manufactured by Revvity
The Cellometer Vision cytometer is a compact and automated cell counting and analysis instrument. It uses image-based cytometry to precisely measure various cellular parameters, such as cell count, viability, and size. The Cellometer Vision provides accurate and reliable data to support research and development activities across multiple scientific disciplines.
Automatically generated - may contain errors
Lab products found in correlation
Giemsa solution, by Merck Group (1 mentions)
Celltiter glo, by Promega (1 mentions)
Knockout dmem, by Thermo Fisher Scientific (1 mentions)
Z 4 hydroxytamoxifen, by Merck Group (1 mentions)
2 mercaptoethanol, by Thermo Fisher Scientific (1 mentions)
Mem non essential amino acids solution, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Glutamax supplement, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
2 protocols using cellometer vision cytometer
1
Proliferation, Viability, and Focus Formation Assays
2
Cre-mediated Sphk1 Knockout in mESCs
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mESCs were grown in a medium of KnockOut DMEM (ThermoFisher, Waltham, MA) with 1% GlutaMAX Supplement (ThermoFisher), 1% penicillin‐streptomycin (GIBCO), 1% modified Eagle's medium (MEM) nonessential amino acids solution (GIBCO), 0.1 mM 2‐mercaptoethanol (GIBCO), 103 units/mL ESGRO Recombinant Mouse LIF Protein (Millipore), and 15% FBS (Sigma). Cells were cultured on irradiated C57BL/6 MEFs (GIBCO) at a density of 2 × 106 cells/10‐cm dish. Cells were counted using a Cellometer Vision cytometer (Nexcelom, Lawrence, MA) as per manufacturer's instructions. To induce Cre‐mediated excision of the Sphk1 gene, (Z)‐4‐hydroxytamoxifen (Sigma) was added at a concentration of 6 μM for 48 hours. For S1P addback experiments, S1P was sonicated in albumin and added to medium at 1 μM.
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