HEK-293 were cultured in DMEM high-glucose (GE Healthcare cat# SH30081.01) containing 10% bovine calf serum (GE Healthcare cat# SH30072.03), 2 mM L-glutamate (Wisent cat# 609065, 100 IU penicillin and 100 µg/ml streptomycin (Wisent cat# 450201). U2OS were cultured in DMEM high-glucose containing 10% tetracyclin-free fetal bovine serum (FBS) (Wisent cat# 081150) 2 mM L-glutamate, 100 IU penicillin and 100 g/ml streptomycin. Tetracyclin-free FBS was used to limit Cas9 expression. U2OS were starved for 2 hr in Earle’s balanced salts medium (Sigma cat# E2888).
L glutamate
Manufactured by Wisent
L-glutamate is a chemical compound that serves as a core component in various laboratory equipment and assays. It functions as an essential amino acid and plays a crucial role in various biological processes. The specific applications and intended uses of L-glutamate in laboratory settings may vary depending on the specific context and requirements of the research or analysis being conducted.
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6 protocols using l glutamate
1
Culturing HEK-293 and U2OS cells
2
Isolation and Culture of Murine Bone Marrow Macrophages
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Bone marrow cells were isolated from femurs of around 12 weeks old mice, as described elsewhere (Trouplin et al., 2013 (link)). Cells were seeded in bacterial dishes and cultured in DMEM high-glucose (GE Healthcare cat# SH30081.01) containing 10% heat inactivated fetal calf serum (FBS; 080–150), 2 mM L-glutamate (Wisent cat# 609065, 100 IU penicillin and 100 µg/ml streptomycin (Wisent cat# 450201) and containing 20% L929 cell-conditioned medium as a source of M-CSF. After 7 days, cells were harvested using cold PBS, seeded, and kept in DMEM, 10% FBS and penicillin/streptomycin for 24 hr before experiment.
3
Cell Culture Media Composition
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All cell lines were cultured in Dulbecco’s Modified Eagle’s medium (DMEM) high-glucose (GE Healthcare, catalog no. SH30081.01) containing 10% bovine calf serum (GE Healthcare, catalog no. SH30072.03), 2 mM l -glutamate (Wisent, catalog no. 609065), 100 IU of penicillin, and streptomycin (100 μg/ml) (Wisent, catalog no. 450201). Serum starvation media are as follows: DMEM high-glucose containing 2 mM l -glutamate, 100 IU of penicillin, and streptomycin (100 μg/ml). Cell lines were routinely tested for mycoplasma contamination using the mycoplasma detection kit (biotool, catalog no. B39038).
4
Huh-7.5 and miR-122 KO cell culture
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Huh-7.5 cells were kindly provided by C. M. Rice and miR-122 KO Huh-7.5 cells were kindly provided by M. Evans (24 (link), 40 (link)). Both Huh-7.5 and miR-122 KO cells were maintained in Dulbecco’s minimal essential medium supplemented with 10% fetal bovine serum, 1% nonessential amino acids, and 200 µM L-glutamate (Wisent, Inc.).
5
Cell Culture Maintenance and Mycoplasma Testing
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Cell lines were cultured in DMEM high-glucose (GE Healthcare cat# SH30081.01) containing 10% bovine calf serum (GE Healthcare cat# SH30072.03), 2 mM L-glutamate (Wisent cat# 609065), 100 IU penicillin and 100 µg/ml streptomycin (Wisent cat# 450201). Serum starvation media: DMEM high-glucose containing 2 mM L-glutamate, 100 IU penicillin and 100 µg/ml streptomycin. Cell lines were tested for mycoplasma contamination routinely using the mycoplasma detection kit (Lonza; cat# LT07-318). Earle′s Balanced Salts (EBSS) was purchased from Sigma (E2888).
6
Cell Culture Maintenance Protocol
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All cells were cultured in Dulbecco's modified Eagle's medium (DMEM) high-glucose (GE Healthcare cat# SH30081.01) containing 10% bovine calf serum (GE Healthcare cat# SH30072.03), 2-mM L-glutamate (Wisent cat# 609065), 100 IU penicillin, and 100 μg/ml streptomycin (Wisent cat# 450201). Cell lines were checked monthly for Mycoplasma contamination using the Mycoplasma detection kit (biotool cat# B39038).
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