IMR-32 human neuroblastoma cell line was cultured in EMEM medium (M4655, Sigma-Aldrich) supplemented with 10% fetal calf serum (10270106, Gibco), 1% non-essential amino acid solution (M7145, Sigma-Aldrich), 1 mM sodium pyruvate (S8636, Sigma-Aldrich) and 50 µg/ml gentamicin (G1397, Sigma-Aldrich). CHP-134 cells were grown in RPMI 1640 medium (R8758, Sigma-Aldrich) supplemented with 10% FCS and 50 µg/ml gentamicin. LAN-1 cells were cultured in EMEM/F-12 (N6658, Sigma-Aldrich) medium diluted in 1:1 ratio, supplemented with 10% FCS, 1% non-essential amino acid solution, 1 mM sodium pyruvate and 50 µg/ml gentamicin, while LAN-5 cells in RPMI 1640 medium supplemented with 20% FCS and 50 µg/ml gentamicin. For preparation of positive controls, IMR-32 and CHP-134 cells were cultured in amino acids-deprived Earle’s Balanced Salt (E2888, Sigma-Aldrich), supplemented with 10% FCS for 24 h. All cell lines were grown at 37 °C in a 5% CO2 atmosphere.
E2888
Manufactured by Merck Group
The E2888 is a laboratory equipment product manufactured by Merck Group. It serves as a versatile instrument for various scientific applications. The core function of the E2888 is to facilitate efficient and accurate measurements and analyses within a laboratory setting. Detailed specifications and technical details are available upon request.
Automatically generated - may contain errors
Lab products found in correlation
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (4 mentions)
M7145, by Merck Group (3 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (2 mentions)
B 6766, by Merck Group (2 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
Synergy ht plate reader, by Agilent Technologies (2 mentions)
Hyclone cosmic calf serum, by Cytiva (2 mentions)
Prism 7, by GraphPad (2 mentions)
Chemidoc imaging system, by Bio-Rad (2 mentions)
S8636, by Merck Group (1 mentions)
R8758, by Merck Group (1 mentions)
Emem f 12, by Merck Group (1 mentions)
M4655, by Merck Group (1 mentions)
Rpmi 1640 medium, by Merck Group (1 mentions)
Gentamicin, by Merck Group (1 mentions)
E3024, by Merck Group (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
D6546, by Merck Group (1 mentions)
B1793, by Merck Group (1 mentions)
Hy 17589a, by MedChemExpress (1 mentions)
13 protocols using e2888
1
Culturing Human Neuroblastoma Cell Lines
2
MCF-7 and MDA-MB-231 Cell Assays with AKT Activation
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Cell culture of standard MCF-7 and MDA-MB-231 cells as well as transient transfection assays were performed essentially as described previously (Dardenne et al. 2012 (link); Samaan et al. 2014 (link)). Sequences of siRNAs and TOSS are provided in Supplemental Table S1 . AKT activation experiments were performed as follows: 24 h after siRNA transfection, cells were first starved in serum-free medium (Earle's Balanced Salts with Sodium medium, Sigma E3024 and E2888) for 16 h and then reactivated in medium containing 5 µg/mL of SC79 (pan-AKT activator by phosphorylation; S7863, Selleckchem) for 1 h.
3
Cell Culture and Starvation Protocol
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Cells were cultured in DMEM (D6546; Sigma-Aldrich) supplemented with 10% FBS (172012; Sigma-Aldrich), 50 U/ml penicillin, 50 µg/ml streptomycin (15070-063; Gibco), and 2 mM glutamine (25030-081; Gibco; regular medium) in a 5% CO2 incubator. For starvation treatment, cells were washed twice with PBS and cultured in Earle’s balanced salt solution (E2888; Sigma-Aldrich) or amino acid–free DMEM (048-33575; Wako) without FBS. For bafilomycin A1 treatment (B1793; Sigma-Aldrich), cells were cultured with 100 nM bafilomycin A1 for 2 h.
4
Rabbit Genome Editing Methodology
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Methods of rabbit genome editing have been described previously in detail.37 (link) Briefly, pronuclear stage rabbit embryos were injected with approximately 2 to 5 pl RNase-free TE buffer (1 mM Tris-Cl, pH 8.0, 0.1 mM EDTA) containing 150 ng/µL Cas9 mRNA and 50 ng/µL sgRNA. Injected embryos were washed three times in embryo culture medium, which consisted of Earle's Balanced Salt Solution (E2888, Sigma, St Louis, MO) supplemented with nonessential amino acids (M7145, Sigma), essential amino acids (B-6766, Sigma), 1 mM l -glutamine (25030-081, Life Technologies, Grand Island, NY), 0.4 mM sodium pyruvate (11360–070, Life Technologies), and 10% fetal bovine serum. Twenty to thirty embryos were transferred surgically to oviducts of each synchronized recipient doe. For gRNA validation, instead of transferring to recipients, the injected embryos were washed and cultured in the medium at 38.5 °C in 5% CO2 for additional 3 days until they reach blastocyst stage.
5
Manipulation of Cellular Stress Pathways
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HEK293T, HeLa, U2OS, Vero-E6, and p62 KO Vero-E6 cells were cultured in Dulbecco’s modified Eagle’s medium (11995065, Gibco) supplemented with 10% fetal bovine serum (12303C, Sigma-Aldrich) and 1% penicillin-streptomycin (15140163, Gibco) at 37°C with 5% CO2. CRISPR-Cas9 was used to generate p62 KO Vero cell line. For starvation treatment, cells were washed twice with phosphate-buffered saline (SH30256.01, Hyclone) and cultured in Earle’s balanced salt solution (E2888, Sigma-Aldrich) for 12 h. To induce ER stress, CPA (GC10268, GLPBIO) and thapsigargin (12758, Cell Signaling Technology) were used to treat cells for 12h or 6h, respectively. For chloroquine treatment, cells were incubated with 100 μM chloroquine (HY-17589A, MedChemExpress) for different times. For 1,6-hexanediol treatment, cells were washed with PBS for twice and incubated with 3% 1,6- hexanediol (H810887, Macklin) for 1 min, and then fixed in 4% PFA for immunofluorescence or lysed in TAP lysis buffer for subsequent immunoblotting. All cells were tested for mycoplasma negative.
6
Induction of Host Defense and Autophagy Signaling
7
Inducing Autophagy in Melanoma Cells
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The human malignant melanoma cell lines, A375, SK-MEL-28, and SK-MEL-5 cell lines were obtained from American Type Culture Collection (ATCC) and were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Biological Industries) supplemented with 10% fetal bovine serum (FBS; Biological Industries) and 1% penicillin and streptomycin (Biological Industries) at 37 °C in 5% CO2 (v/v). To induce autophagy, cells were incubated with the starvation medium Earle’s Balanced Salt Solution (EBSS; E2888, Sigma-Aldrich) instead of the normal medium for 24 h.
8
Crystal Violet Assay for Cell Viability
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Cells were seeded at 80,000 cells/well in 12-well plates and 24 hours later washed with PBS and media were changed to Earle's Balanced Salt Solution (EBSS, E2888, Sigma-Aldrich). After 24, 48, or 72 hours, EBSS was removed and replaced with DMEM (11965118, Gibco) supplemented with 1% penicillin–streptomycin (5,000 U/mL, 15070063, Gibco) and 10% Hyclone Cosmic Calf Serum (SH3008703, Cytiva). For experiments involving drug treatments, drugs were added at the indicated dose(s) and maintained in the culture medium throughout the experiment 24 hours after seeding. Cells were fixed and stained with 500 µL crystal violet solution (0.5%) for 15 minutes after recovery in replenished medium for 72 hours. Cells were washed with deionized water and plates allowed to dry before images were taken with the Bio-Rad Chemidoc Imaging System. Stain was removed from cells by adding 1 mL of 10% acetic acid/well and plates were rocked for 20 minutes at room temperature. Samples from each well were transferred to a 96-well plate, and 590 nmol/L absorbance was read on a Synergy HT plate reader (BioTek Instruments). Statistical analysis of 590 nmol/L absorbance was performed using a two-way ANOVA followed by Tukey's multi-comparison test in Prism 7 (GraphPad).
9
Studying Amino Acid and Glucose Starvation Effects
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For amino acid starvation, we used EBSS (E2888 from Sigma or SH3002902 from GE Healthcare Life Sciences) supplemented with 10% dialyzed fetal bovine serum (dFBS) (ThermoFisher Scientific, 26400044). Cells were pre-treated with chemical agents 30 minutes before they were incubated with amino acid starvation medium for 1 h to analyze ULK1 activity or interaction. For glucose starvation, we used DMEM (ThermoFisher Scientific, 11966025) supplemented with 10% dFBS. Cells were incubated with glucose starvation medium for 2 h or indicated periods of time before cells were treated with chemicals or mTORC1 inhibiting conditions. All the chemicals used were resolved in dimethyl sulfoxide (DMSO) as stock and used at the indicated concentrations for each experiment. Mitochondrial inhibitors were treated to cells in full medium at the indicated concentrations for 30 min before cells were starved of amino acids otherwise specified. All the chemicals for analysis of their effects on ULK1 activity, Vps34 activity and autophagy were present not only in pre-incubation but also during starvation or mTORC1 inhibiting treatments.
10
Monitoring Autophagy via Fluorescent-Tagged LC3
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A tandem fluorescent-tagged LC3 construct was used to monitor the formation of GFP-LC3 and RFP-LC3 punctae [12 (link)]. Plasmids were transfected into H1299 and PC-9 cells using the Lipofectamine® RNAiMAX Reagent (Invitrogen). After 48 h, cells were washed with PBS and incubated with Earle's balanced salt solution (E2888, Sigma) for an appropriate period. The formation of fluorescent punctae was visualized by confocal microscopy.
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