HeLa cells expressing C1–GFP38 (link) were cultured at 37 °C, 5% CO2 in DMEM (Invitrogen), 10% FCS and 1% penicillin/streptomycin. For microscopy analysis, cells were plated on round 170-nm-thick glass coverslips (Warner Instruments). For live-cell analysis, coverslips with cells were mounted in Rose chambers, which were perfused with complete CO2-independent medium (Invitrogen). To express Plk1TD–RFP, cells were treated with 1 μg ml−1 doxycycline (dox, Sigma).
Complete co2 independent medium
Manufactured by Thermo Fisher Scientific
Complete CO2-independent medium is a cell culture medium formulated to maintain optimal cell growth and proliferation without the need for a CO2 incubator. It is designed to maintain pH and nutrient levels in the absence of a CO2-enriched environment.
Automatically generated - may contain errors
Lab products found in correlation
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Doxycycline dox, by Merck Group (1 mentions)
Cholera toxin, by Merck Group (1 mentions)
Pegfp n1 vector, by Takara Bio (1 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (1 mentions)
Epidermal growth factor (egf), by Merck Group (1 mentions)
Horse serum, by Thermo Fisher Scientific (1 mentions)
Hydrocortisone, by Merck Group (1 mentions)
2 protocols using complete co2 independent medium
1
HeLa Cells Expressing C1-GFP38 Microscopy
2
Centrin-GFP Expressing Cell Lines
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HeLa, U2OS, and RPE-1 cells with cytomegalovirus-driven constitutive expression of Centrin-GFP were described previously (Piel et al., 2000 (link); Lončarek et al., 2008 (link), 2010 (link)). In brief, pEGFP-N1 vector (Takara Bio Inc.) was used to express a full-length Centrin-GFP cDNA in HeLa cells, and Lentilox3.1 vector was used for Centrin-GFP expression in RPE-1 and U2OS cells. The cells were cultured in DMEM (Invitrogen) supplemented with 10% fetal calf serum and 1% penicillin/streptomycin. MCF10A cells (ATCC) were maintained in DMEM supplemented with 0.1 µg/ml cholera toxin (EMD Millipore), 0.5 µg/ml hydrocortisone (Sigma-Aldrich), 10 µg/ml insulin, 0.02 µg/ml EGF (EMD Millipore), 5% horse serum (Invitrogen), and 1% penicillin/streptomycin. All cells were grown at 37°C in a humidified atmosphere of 5% CO2. For live-cell imaging, cells grown on the coverslips were assembled into Rose chambers and perfused with complete CO2-independent medium (Invitrogen).
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