All cell lines were authenticated by STR analysis and were regularly tested for mycoplasma using the MycoAlert Mycoplasma Detection Kit (Lonza). Cells were grown in human plasma-like medium according to the published formulation (Cantor et al., 2017) with 5% dialyzed FBS (Sigma) and Pen/Strep (Invitrogen). Media was changed at least every two days. As needed, cells were incubated in RPMI media (R9010-01, US Biological Life Sciences) with or without serine and glycine with 5% dialyzed FBS. Cells were counted using a Z1 Coulter Particle Counter (Beckman Coulter) and growth rates were calculated using the following formula: growth rate = ln(final cell number/initial cell number)/time.
Rpmi media
Manufactured by US Biological
RPMI media is a common cell culture medium used for the growth and maintenance of a variety of cell types, including mammalian cells, lymphocytes, and hybridomas. It provides the necessary nutrients and growth factors for cell proliferation and survival.
Automatically generated - may contain errors
Lab products found in correlation
Pen strep, by Thermo Fisher Scientific (3 mentions)
Fetal bovine serum (fbs), by Merck Group (3 mentions)
Z1 coulter particle counter, by Beckman Coulter (3 mentions)
Mycoalert mycoplasma detection kit, by Lonza (3 mentions)
Fetal bovine serum (fbs), by US Biological (2 mentions)
Hcc1500, by American Type Culture Collection (2 mentions)
Pen strep, by US Biological (2 mentions)
Fugene 6 transfection reagent, by Promega (2 mentions)
Opti mem, by Thermo Fisher Scientific (2 mentions)
Hek293ft, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin supplement, by Thermo Fisher Scientific (2 mentions)
Mem amino acid supplement, by Thermo Fisher Scientific (2 mentions)
Rnaimax, by Thermo Fisher Scientific (1 mentions)
5 protocols using rpmi media
1
Cell growth rate quantification
2
Culture of Breast Cancer Cell Lines
3
Culture of Breast Cancer Cell Lines
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Hec1A, AGS, and HCC1500 cells were acquired from ATCC. All other cell lines (HCC1806, SUM149, BT549, HCC1937, HCC70, BT20, MCF7, MDAMB453, EFM19, ZR75-1, and T47D) were acquired from the Brugge Lab at Harvard Medical School, and were authenticated by STR analysis. All cell lines were of female origin. Cell lines were regularly tested for mycoplasma using the MycoAlert Mycoplasma Detection Kit (Lonza). Cells were grown in human plasma-like medium according to the published formulation (Cantor et al., 2017 (link)) with 5% dialyzed FBS (Sigma) and Pen/Strep (Invitrogen) at 37 degrees C with 5% CO2. Media was changed at least every two days. As needed, cells were incubated in RPMI media (R9010-01, US Biological Life Sciences) with or without serine and glycine with 5% dialyzed FBS and Pen/Strep. Cells were counted using a Z1 Coulter Particle Counter (Beckman Coulter) and growth rates were calculated using the following formula: growth rate = ln(final cell number/initial cell number)/time.
4
Cell Culture Conditions and Transfection
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HEK293FT (Life Technologies, Carlsbad, CA) and HeLa M cells (provided by P. De Camilli, Yale University, New Haven, CT) were grown in high-glucose DMEM (+l -glutamine), 10% fetal bovine serum, and 1% penicillin/streptomycin supplement (Invitrogen, Carlsbad, CA). Where indicated, cells were starved for 90 min by incubation with amino acid–free RPMI media (US Biological, Swampscott, MA). Amino acid refeeding was performed with 1× MEM amino acid supplement (Invitrogen) added to starvation medium for 15 min. Transfections were performed with 500 ng of DNA, 100 μl of OptiMEM (Invitrogen), and 1.5 μl of FuGENE 6 transfection reagent (Promega, Madison, WI) per 35-mm dish and scaled up proportionately for larger and smaller dishes. siRNA transfections were performed with 5 μl of RNAiMAX (Invitrogen), 500 μl of OptiMEM (Invitrogen), and 7.5 μl of 20 μM siRNA stock suspended in 100 mM KAc and 30 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, pH 7.5. We used 250,000 HEK 293FT cells per 35-mm dish for siRNA transfection and incubated them for 2 d posttransfection before experiments. siRNA sequences purchased from Integrated DNA Technologies (IDT, Coralville, IA) are summarized in Supplemental Table S1.
5
Cell Culture and Transfection Protocol
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HEK293FT (Life Technologies, Carlsbad, CA) and HeLa cells (provided by P. De Camilli, Yale University, New Haven, CT) were grown in high-glucose DMEM (+l -glutamine), 10% fetal bovine serum (FBS), and 1% penicillin/streptomycin supplement (Invitrogen, Carlsbad, CA). Where indicated, cells were starved for 90 min by incubation with amino acid–free Roswell Park Memorial Institute (RPMI) media (US Biological, Swampscott, MA). Amino acid refeeding was performed with 1 × MEM amino acid supplement (Invitrogen) added to starvation medium for 15 min. For starvation/refeeding assays in Figure 5 , 500,000 HeLa cells were seeded in 10-cm dishes for 24 h prior to the start of the experiment. Transfections were performed with 500 ng of DNA, 100 μl of OptiMEM (Invitrogen), and 1.5 μl of FuGENE 6 transfection reagent (Promega, Madison, WI) per 35-mm dish and scaled up proportionately for larger and smaller dishes. For transient transfections, cells were analyzed 2 d posttransfection.
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