H1299 cells (ATCC) were cultured in RPMI (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. Cells were trypsinized and plated at 60–80% confluency on a glass-bottomed imaging dish. The EGFP and p53-GFP plasmids are commercially available through Addgene while the p53-R175H-GFP plasmid was kindly constructed and provided by Dr. Lee Bardwell and Dr. Jane Bardwell in the Department of Developmental and Cell Biology at the University of California, Irvine. Lipofectamine 3000 (Invitrogen, Carlsbad, CA) was used to transfect according to manufacturer’s instructions. Cells were incubated at 37°C and 5% CO2 for 12–24 hours prior to imaging. To induce intra-strand crosslinks, cells were treated with 20 μM cis-diamminedichloroplatinum (II) (cisplatin) (Sigma-Aldrich, St. Louis, MO) for the duration of the experiments. For inhibition of oxidative phosphorylation, cells were treated with 10 μM rotenone and 10 μM antimycin A (Sigma-Aldrich, St. Louis, MO) in 0.35% DMSO for 1 hour prior to imaging. For inhibition of glycolysis, cells were treated with 20 mM 2-deoxy-D-glucose and 5 mM dichloroacetate (Sigma-Aldrich, St. Louis, MO) for 6 hours prior to imaging.
H1299 cells
Manufactured by Thermo Fisher Scientific
Sourced in United States
The H1299 cell line is a non-small cell lung cancer (NSCLC) cell line derived from a human lung carcinoma. It is commonly used in cancer research and drug development studies.
Automatically generated - may contain errors
Lab products found in correlation
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (4 mentions)
H1299 cells, by American Type Culture Collection (2 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (2 mentions)
Dichloroacetate, by Merck Group (1 mentions)
Cisplatin cis diamminedichloroplatinum 2, by Merck Group (1 mentions)
2 deoxy d glucose, by Merck Group (1 mentions)
Antimycin a, by Merck Group (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Roswell park memorial institute (rpmi), by Thermo Fisher Scientific (1 mentions)
Rotenone, by Merck Group (1 mentions)
Improm iitm reverse transcription system kit, by Promega (1 mentions)
Sybr green qpcr supermix, by Thermo Fisher Scientific (1 mentions)
Antibiotic antimycotic solution, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Small interfering rna sirna, by Merck Group (1 mentions)
Hepg2, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Rneasy plus mini kit, by Qiagen (1 mentions)
Hepg2, by American Type Culture Collection (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
6 protocols using h1299 cells
1
Assessing p53 Dynamics in H1299 Cells
2
RNA Extraction and qPCR Analysis of A549 and H1299 Cell Lines
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Using the TRIzol reagent, the total RNA of A549 and H1299 cells was extracted (Invitrogen, Carlsbad, CA, USA). Using the ImProm-IITM Reverse Transcription System kit (Promega Corporation, Madison, WI, USA) and following the manufacturer's instructions, reverse transcribed RNA into cDNA. They then used SYBR® GREEN qPCR Super Mix to perform qPCR (Invitrogen, Carlsbad, CA, USA). To put it briefly, the reaction system was loaded with primer, cDNA, and SYBR GREEN qPCR Super Mix. After two minutes of denaturation at 95°C, the PCR thermocycling parameters were as follows: forty cycles of 15 seconds at 95°C and 32 seconds at 60°C. The internal control used to normalize BLK expression was GAPDH. BLK expression was evaluated using the 2 -ΔΔCt technique. The primers were: GAPDH forward, 5′-CCAGGGAAGGATGTAGCTGTG -3′ and reverse, 5′-GGCTGTTGTCATACTTCTCATGG -3′; DLEU2 forward, 5′-TCCGAGAGTATAGCGCCACT -3′ and reverse, 5′-ACTGCCCTTTGCTCCAAGTA -3′. Every group underwent three rounds of experiments.
3
Cell Culture Protocols for H1299 and Expi293
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H1299 cells were purchased from the American Type Culture Collection (Manassas, VA, USA). H1299 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% (v/v) fetal bovine serum (FBS), 100 IU/mL penicillin and 100 µg/mL streptomycin. Cells were grown in a humidified 37 °C incubator with 5% CO2.
Expi293 cells were acquired from Union-Biotech (Shanghai, China) and maintained in shaking incubators at 37 °C, humid atmosphere and 8% CO2 in a serum-free proprietary culture medium (Union-293 UP1000, chemically defined medium with L-glutamate).
Expi293 cells were acquired from Union-Biotech (Shanghai, China) and maintained in shaking incubators at 37 °C, humid atmosphere and 8% CO2 in a serum-free proprietary culture medium (Union-293 UP1000, chemically defined medium with L-glutamate).
4
Culturing H1299 Lung Cancer Cells
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H1299 cells (non-small cell lung carcinoma, ATCC; Manassas, VA, USA; Cat# CRL-5803) were cultured in DMEM (Gibco; Waltham, MA, USA; Cat# 12100-61) supplemented with 10% FBS (Gibco; Waltham, MA, USA; Cat# A4766801) and Antibiotic-Antimycotic solution (Gibco; Waltham, MA, USA; Cat# 15240-062). H1299 cell lines tested negative for mycoplasma and were used at passage 20 or lower. E12−/− H1299 cell lines were generated as described previously [37 (link)].
5
Cell Culture and siRNA Transfection
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H1299 and HepG2 cells were purchased from the American Type Culture Collection. H1299 cells were cultured in RPMI‐1640 (Gibco) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. HepG2 cells were cultured in DMEM (Gibco) supplemented with 10% FBS and 1% penicillin/streptomycin. All cells were cultured at 37°C with 5% CO2. Small‐interfering RNA (siRNA) was manufactured by Sigma‐Aldrich. The sequences are listed in Table S1 . Lipofectamine RNAiMAX (Thermo Fisher Scientific) was used for transfection of siRNA. Total RNA was extracted at 48 h after treatment for reverse‐transcription quantitative PCR (RT‐qPCR). RNA extraction was performed with an RNeasy Plus Mini kit (Qiagen) according to the manufacturer's instructions.
6
Silencing COL5A2 and EPHB2 in LUAD Cells
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The human LUAD cell lines, including A549 cells and H1299 cells, were purchased from the American Type Culture Collection (ATCC) (Manassas, VA, USA). The A549 and H1299 cells were cultured in DMEM (Gibco, NYC, USA) containing 10% fetal bovine serum (FBS) (Gibco, NYC, USA) and 100 U/ml penicillin–streptomycin (Gibco, NYC, USA) in an incubator with 5% CO2 at 37 °C. The cells were transfected with 50 nM COL5A2 siRNA or EPHB2 siRNA in basic DMEM for 6–8 h and then changed to DMEM supplemented with 10% FBS and 100 U/ml penicillin–streptomycin. The A549 and H1299 cells were treated with COL5A2 siRNA or EPHB2 siRNA for 48 h for the next experiment.
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