hTERT-RPE1 cells were obtained from the American Type Culture Collection (ATCC CRL-4000) and grown in Dulbecco's modified Eagle's medium (DMEM)/F-12, GlutaMAX medium (Gibco) supplemented with 10% (v/v) fetal bovine serum (Gibco), 200 μg/ml Hygromycin B, 50 U/ml penicillin, and 50 μg/ml streptomycin (GE Healthcare) at 37 °C and 5% CO2. U2OS, 293, and 293T cells were obtained from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures (DSMZ no. ACC 785, ACC 305, and ACC 635, respectively) and grown in DMEM, GlutaMAX medium (Gibco) supplemented with 10% (v/v) fetal bovine serum (Gibco), and 50 U/ml penicillin and 50 μg/ml streptomycin (GE Healthcare) at 37 °C and 5 % CO2. PCR-based Mycoplasma contamination tests were regularly performed using the VenorGeM Classic kit (Minerva Biolabs). Autophagy was induced by amino acid starvation using EBSS medium (Gibco).
Ebss medium
Manufactured by Thermo Fisher Scientific
EBSS medium is a balanced salt solution designed for use in cell and tissue culture applications. It provides a buffered, isotonic environment to maintain the pH and osmolarity required for optimal cell growth and viability.
Automatically generated - may contain errors
Lab products found in correlation
Glutamax medium, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Penicillin, by GE Healthcare (1 mentions)
Streptomycin, by GE Healthcare (1 mentions)
Venor gem classic kit, by Minerva Biolabs (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Lookout mycoplasma detection kit, by Merck Group (1 mentions)
4 protocols using ebss medium
1
Cultivation and Autophagy Induction in Cell Lines
2
In vivo and ex vivo mitochondrial analysis
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For in vivo mitochondrial network analysis, mice infected with Ad-Cox8-GFP were perfused with 10% formalin. Liver samples were further processed in 30% sucrose solution and embedded in OCT for cryosectioning. For ex vivo mitochondrial network analysis, hepatocytes seeded on coverslips were infected with Ad-Cox8-mCherry for 8 hours, followed by a second virus infection for another 40 hours. Cells were then switched to the EBSS medium (Gibco, 14155063) containing 0.5 mM MgCl2, 1.8 mM CaCl2, and 1 g/L glucose for 4 hours and fixed with 4% paraformaldehyde for 10 minutes. Slides were mounted with mounting media containing DAPI to stain nuclei. All images were acquired by confocal microscopy and analyzed by ImageJ software (NIH). The image of GFP channel is subjected to default “Moments_Thresholding” to measure mitochondrial area and perimeter.
3
Autophagic Regulation in Glial and Neuronal Cells
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BV2 shCtrl and shAtg7 cells, glioma C6 (ATCC® CCL-107™) and neuronal MN9D (gift from Dr. Alfred Heller, University of Chicago, USA) rodent cell lines have been used in this study. BV2 and C6 cells were cultured in complete DMEM Glutamax (Gibco, 61965059) with 10% fetal bovine serum (Gibco, 10270-106, LOT 42F1490K) and 1% penicillin/streptomycin (Gibco, 15140122). MN9D cells were cultured in DMEM/F12 (Gibco, 31330038) with 10% fetal bovine serum and 1% penicillin/streptomycin. Cells were regularly tested with LookOut mycoplasma detection kit (Sigma, MP0035). BV2 cells were treated with either 100 ng/ml LPS (likewise MN9D cells, in the coculture experiment), 10 ng/ml IL-4, or 250 nM Torin1 for the indicated time points. As control, cells were treated with the respective reagent’s solvent. BV2 cells were starved using Earle’s Balanced Salt Solution (EBSS medium, Gibco, 24010043). Co-treatment with 40 nM BafA1, 2 h before sample collection, was used to block autophagic flux.
4
Long-Term Autophagy Induction Protocol
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U1810, HeLa or MEF cells were seeded at 75,000 cells per well in 6-well plates in complete culture medium (as described above). One-day post-seeding, culture medium was removed, cells were washed twice with PBS (Gibco, SH300028.02) and treated with either DMSO ([Sigma-Aldrich, D4540]; 1:4000, used as control) or one of the following autophagy inducers: 250 nM torin1, 50 μM carbamazepine, 100 mM trehalose or starved using Earle’s Balanced Salt Solution (EBSS medium; Gibco, 24,010–043). After 4 h, cells were collected and transferred into T75 flasks with fresh complete cell culture medium. Thereafter, cells were cultivated for a period of time, ranging from 1 to 4 weeks before experiments were performed. Cells were split 3 times per week during this recovery period. Before performing the experiments, cell number was normalized. An illustrated description of the method is shown in Fig. S1A.
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