HCT116 p53+/+ and HCT116 p53−/− cells were cultured in Dulbecco's modified Eagle medium (DMEM, Corning) supplemented with 10% fetal bovine serum (FBS, Gemini Bio-Products), 100 units/mL of penicillin, and 100 µg/mL of streptomycin (Gemini Bio-Products). These cells have been generated previously42 (link). EB3, SUPT1, DB (purchased from ATTC) and DOHH2 (purchased from DSMZ) lymphoma cell lines were cultured in RPMI 1640 medium (Corning) with 10% FBS, 100 units/mL of penicillin, and 100 µg/mL of streptomycin (Gemini Bio-Products). CA46 (purchased from ATTC) and LY3 (purchased from DSMZ) cells were cultured in RPMI medium with 20% FBS. All cells were cultured at 37°C with 5% CO2. All cells were routinely tested for mycoplasma contamination using MycoAlert™ Mycoplasma Detection Kit (Lonza). L-DON, BPTES and camptothecin were purchased from Sigma. Docetaxel was purchased from Selleckchem, and Compound 968 was purchased from Calbiochem.
Streptomycin
Manufactured by Gemini Bio
Sourced in United States, Canada
Streptomycin is a broad-spectrum antibiotic produced by the bacterium Streptomyces griseus. It is a protein synthesis inhibitor that works by binding to the 30S subunit of the bacterial ribosome, preventing the initiation of translation.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin, by Gemini Bio (73 mentions)
Fetal bovine serum (fbs), by Gemini Bio (22 mentions)
L glutamine, by Gemini Bio (15 mentions)
Dulbecco modified eagle medium (dmem), by Corning (10 mentions)
Fungizone, by Gemini Bio (7 mentions)
Penicillin, by Thermo Fisher Scientific (6 mentions)
Streptomycin, by Thermo Fisher Scientific (6 mentions)
β mercaptoethanol, by Merck Group (5 mentions)
Hepes, by Corning (5 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (5 mentions)
Sodium pyruvate, by GE Healthcare (5 mentions)
Penicillin g, by Gemini Bio (4 mentions)
Glutamine, by Gemini Bio (4 mentions)
Gentamycin, by Avantor (4 mentions)
Gene pulser xcell system, by Bio-Rad (4 mentions)
α mem, by Thermo Fisher Scientific (4 mentions)
Rpmi 1640, by Corning (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (4 mentions)
Doxycycline, by Merck Group (3 mentions)
83 protocols using streptomycin
1
Cell Culture and Compound Screening
2
CRISPR-Cas9 Knockout of Orai and STIM Proteins
3
Transduction of CD34+ Hematopoietic Stem Cells
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PB CD34+ samples were obtained from healthy donors from plerixafor and G-CSF mobilization. Cells were thawed and plated at 1 × 106 cells/mL on non-tissue culture-treated 96-well plates pre-coated with retronectin (20 μg/mL; Takara Shuzo). Cells were pre-stimulated for 24 h in X-Vivo 15 medium (Lonza) supplemented with 1× glutamine, penicillin, and streptomycin (Gemini Bio-Products), human Flt-3 ligand (50 ng/mL), human SCF (50 ng/mL), human TPO (50 ng/mL), and human IL-3 (20 ng/mL) (cytokines: PeproTech). CD34+ cells were transduced with concentrated viral supernatants at transduction concentrations of 2 × 105 TU/mL, 6 × 105 TU/mL, 2 × 106 TU/mL, and 6 × 106 TU/mL with transduction enhancer Poloxamer 338 (1 mg/mL) (BASF, Ludwigshafen, Germany). At 24 h after transduction, cells were washed and plated under myeloid culture conditions. Cells were cultured for 2 weeks after transduction in basal bone marrow media consisting of Iscove-modified Dulbecco’s medium (Lonza) 1× glutamine, penicillin, and streptomycin (Gemini Bio-Products), 20% fetal bovine serum, and 0.52% BSA (Sigma-Aldrich) supplemented with human SCF (25 ng/mL), human IL-3 (5 ng/mL), and human IL-6 (10 ng/mL).
4
Cell Culture and Compound Screening
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HCT116 p53+/+ and HCT116 p53−/− cells were cultured in Dulbecco's modified Eagle medium (DMEM, Corning) supplemented with 10% fetal bovine serum (FBS, Gemini Bio-Products), 100 units/mL of penicillin, and 100 µg/mL of streptomycin (Gemini Bio-Products). These cells have been generated previously42 (link). EB3, SUPT1, DB (purchased from ATTC) and DOHH2 (purchased from DSMZ) lymphoma cell lines were cultured in RPMI 1640 medium (Corning) with 10% FBS, 100 units/mL of penicillin, and 100 µg/mL of streptomycin (Gemini Bio-Products). CA46 (purchased from ATTC) and LY3 (purchased from DSMZ) cells were cultured in RPMI medium with 20% FBS. All cells were cultured at 37°C with 5% CO2. All cells were routinely tested for mycoplasma contamination using MycoAlert™ Mycoplasma Detection Kit (Lonza). L-DON, BPTES and camptothecin were purchased from Sigma. Docetaxel was purchased from Selleckchem, and Compound 968 was purchased from Calbiochem.
5
CRISPR-Cas9 Knockout of Orai and STIM Proteins
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A majority of the experiments were carried out on HEK293 cells, referred to in this manuscript as “HEK.” HEK-Orai1/2/3TKOand HEK-STIM1/2DKO lines cells were generated using the CRISPR-Cas9 nickase system (Yoast et al., 2020 (link); Zhou et al., 2018a (link)), and cultured in DMEM (Corning Cellgro) supplemented with 10% fetal bovine serum, plasmocin (InvivoGen), penicillin and streptomycin (Gemini Bioproducts) at 37°C with 5% CO2. Additional physiological experiments were performed on HeLa cells stably expressing GFP-NFAT1 (HeLa-GFP-NFAT1 cells) that were developed earlier (Zhou et al., 2016 (link)), and cultured in the same medium as above, supplemented with G418 (Corning, 100 mg/ml). Further supplemental experiments were carried out on Jurkat T cell lines, which were cultured in RPMI 1640 (Corning Cellgro) supplemented with 10% fetal bovine serum, plasmocin (InvivoGen), penicillin and streptomycin (Gemini Bioproducts) at 37°C with 5% CO2.
6
Culturing NIH/3T3 and HEK293T Cell Lines
7
Characterization of Cytokine-Producing T Cells
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Human PBMCs were rested overnight at 37°C in RPMI 1640 medium (catalog# RP-21, Omega Scientific, Inc.) supplemented with 5% human serum (catalog# 100–512, Gemini Bio-Products), 2 mM L-alanyl-L-glutamine (GlutaMAX-I, catalog# 35050061, Thermo Fisher Scientific), 100 U/ml penicillin, and 100 μg/ml streptomycin (catalog# 400–109, Gemini Bio-Products) and subsequently stimulated with DENV MP (1 μg/ml for individual peptides) or Dynabeads Human T-Activator CD3/CD28 for T Cell Expansion and Activation (catalog# 11132D, Thermo Fisher Scientific) for 3 h at 37°C. IFN-γ- and IL-10-producing cells were labeled using IFN-γ Secretion Assay - Detection Kit (PE), human (catalog# 130–054-202, Miltenyi Biotech) and IL-10 Secretion Assay – Detection Kit (APC), human (catalog# 130–090-761, Miltenyi Biotech), respectively, according to the manufacturer’s instructions. Subsequently, PBMCs were stained with anti-human CD3, CD4, CD8, CD14, CD19, CD45RA, and CCR7 (see Table S4 for antibody details). CD4 IL-10−IFN-γ− DN, IL-10+IFN-γ− IL-10 SP and IL-10−IFN-γ+ IFN-γ SP, and IL-10+IFN-γ+ DP cells were sorted using a FACSAria II cell sorter. Data were analyzed using FlowJo.
8
Cell Culture Conditions and Maintenance
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NIH-3T3 and HEK293T cells were grown in high glucose, pyruvate-supplemented DMEM (Gibco) with 10% fetal bovine serum (FBS), 2 mM glutamine, 10 U/ml penicillin and 10 μg/ml streptomycin (Gemini Bio-Products). NIH-3T3 FlpIn cells (gift from R. Rohatgi) were grown in the same medium supplemented with non-essential amino acids (Gibco). Light-II NIH-3T3 cells24 (link) were grown in the same medium except with 10% bovine calf serum (ATCC), and IMCD3 cells were grown in DMEM/F12 medium (Gibco) with FBS, glutamine, penicillin and streptomycin additives. Serum starvation was done using medium with 0.5% FBS for NIH-3T3 cells, 0.5% calf serum for Light-II NIH-3T3 cells, and 0.2% FBS for IMCD3 cells. IMCD3 FlpIn cells were provided by Peter Jackson. NIH-3T3 cells were obtained from ATCC. HEK239T-EcR-ShhN cells were provided by Philip Beachy. Cells were confirmed to be mycoplasma free with the MycoAlert system (Lonza).
9
Quantifying Nucleic Acid Content
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The Quant-iT ™ Picogreen ® assay kit and the Presto Blue kit were from Life Technologies (Carlsbad, US).
Bovine serum, L-glutamine, penicillin and streptomycin were from Gemini Bio-Products (Sera Laboratories International, Bolney, UK). All other reagents were purchased from Sigma-Aldrich (Poole, UK).
Bovine serum, L-glutamine, penicillin and streptomycin were from Gemini Bio-Products (Sera Laboratories International, Bolney, UK). All other reagents were purchased from Sigma-Aldrich (Poole, UK).
10
Cell Line Characterization and Maintenance
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