Reagents used in the present study included RPMI-1640 (pH 7.2) medium, high-sugar DMEM medium (Beijing Solarbio Science & Technology Co., Ltd.), South American fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.), dimethyl sulfoxide (DMSO), trypsin, penicillin-streptomycin, MTT, DAPI nuclear stain (Beijing Zhongsheng Ruitai Technology Co., Ltd.), 3% glutaraldehyde (Alfa Aesar; Thermo Fisher Scientific, Inc.) mouse Anti-Human CD61 PE-labeled antibody (Beijing Solarbio Science & Technology Co., Ltd.), cisplatin (Qilu Pharmaceutical), 10% sheep serum (NGS; Shanghai Biosun Sci&Tech Co., Ltd.) and 0.05% DAB containing 0.03% hydrogen peroxide (protected from light). The cell endothelial growth factor VEGF (1:100; cat. no. 251659) and apoptosis-related protein caspase-3 (1:100; cat. no. 341034) primary antibodies and the secondary goat anti-mouse antibodies IgG were obtained from Zen BioScience Co., Ltd. (1:200; cat. no. 550047). Primary antibodies against cleaved caspase-3 (1:1,000; cat. no. ab13847), GAPDH (1:1,000; cat. no. ab8245) and goat anti-rabbit horseradish peroxidase secondary antibody (1:3,000; cat. no. ab7621) were obtained from Abcam.
Rpmi 1640
Manufactured by Solarbio
Sourced in China, United States
RPMI-1640 is a commonly used cell culture medium formulated for the growth and maintenance of a variety of cell types, including human and animal cells. It provides the necessary nutrients and conditions to support cell growth and proliferation in a laboratory setting.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (12 mentions)
Fetal bovine serum (fbs), by Solarbio (11 mentions)
Dulbecco modified eagle medium (dmem), by Solarbio (11 mentions)
Penicillin, by Solarbio (9 mentions)
Streptomycin, by Solarbio (9 mentions)
Penicillin streptomycin, by Solarbio (9 mentions)
Rpmi 1640, by Thermo Fisher Scientific (6 mentions)
Dulbecco s modified eagle s medium, by Solarbio (4 mentions)
Fetal bovine serum (fbs), by Sartorius (3 mentions)
Dulbecco s modified eagle medium dmem, by Solarbio (3 mentions)
Phosphate buffered saline (pbs), by Solarbio (3 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
A549 cell, by American Type Culture Collection (2 mentions)
H1299, by American Type Culture Collection (2 mentions)
Hek293t, by American Type Culture Collection (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Dulbecco s modified eagle s medium dmem, by Solarbio (2 mentions)
Humidified incubator, by Thermo Fisher Scientific (2 mentions)
Trypsin edta, by Solarbio (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
86 protocols using rpmi 1640
1
Cell Viability and Apoptosis Assay
2
Cytokine Response to Oral Bacterial Interactions
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dTHP-1 cells were seeded in 6-well plates (1×106 cells/well) and were infected with 0.5 μl/ml LPS (Solarbio, Beijing, China), PBS, the monoculture, the co-culture, and the coaggregates of S. gordonii and F. nucleatum subsp. polymorphum, respectively. After 2 h or 4 h, the plates were washed with PBS and cultured in fresh RPMI 1640 containing 10% FBS, gentamicin (300 μg/ml), and metronidazole (200 μg/ml) (Solarbio, Beijing, China) to kill extracellular bacteria. After 2 h, the plates were cultured in fresh RPMI 1640 containing 10% FBS, gentamicin (60 μg/ml), and metronidazole (40 μg/ml). Supernatants of samples collected at different time points were collected by centrifugation at 3,000 rpm (20 min, 4°C). Pro-inflammatory cytokines IL-1β, IL-6, and TNF-α were analyzed using ELISA kits (Neobioscience, Shenzhen, China) according to the manufacturer’s instructions.
3
Cell Culture Protocols for Cancer Research
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Human prostatic carcinoma cell line PC3, human breast adenocarcinoma cell line MCF-7, human gastric carcinoma cell line MGC-803, human lung carcinoma cell line PC9, normal human prostatic cell line WPMY-1 were all purchased from the Cell Bank of Shanghai Institute of Biochemistry and Cell Biology. They were cultured in RPMI-1640 and DMEM complete medium (Solarbio, China) at 37 °C in a 5% CO2 humidified atmosphere.
4
NHBE Cell Transfection with NLRP3
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Normal human bronchial epithelial (NHBE) cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA), and cultivated in RPMI1640 (Solarbio, Beijing, China) suspended with 10% fetal bovine serum (FBS, Solarbio, Beijing, China), 100 U/mL streptomycin (Solarbio, Beijing, China) and 100 U/mL penicillin (Solarbio, Beijing, China) at 37 °C and 5% CO2.
NHBE cells were suspended into serum-free RPMI1640, plated into 6-well plates (1 × 106 cells/mL per well), and transfected by either pCDNA3.1-NLRP3 vectors (served as the NLRP3 group) or pCDNA3.1 vectors (named the NC group) via using Lipofectamine 3000 (Thermo Fisher Scientific, Shanghai, China). The transfection was implemented following the manufacturer’s instruction. pCDNA3.1-NLRP3 vectors and pCDNA3.1 vectors were all commercially provided by GeneChem (Shanghai, China).
NHBE cells were suspended into serum-free RPMI1640, plated into 6-well plates (1 × 106 cells/mL per well), and transfected by either pCDNA3.1-NLRP3 vectors (served as the NLRP3 group) or pCDNA3.1 vectors (named the NC group) via using Lipofectamine 3000 (Thermo Fisher Scientific, Shanghai, China). The transfection was implemented following the manufacturer’s instruction. pCDNA3.1-NLRP3 vectors and pCDNA3.1 vectors were all commercially provided by GeneChem (Shanghai, China).
5
IL-7 Stimulation of Enriched PBMCs
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Enriched PBMCs were resuspended in RPMI 1640 (Solarbio; Beijing, China), supplemented with 10% FBS. Cells were stimulated with recombinant human IL-7 (50 ng/mL) (Beyotime; Shanghai, China) in the presence or absence of 150 nM tofacitinib (Meilun Biotechnology; Dalian, China) and cultured for up to 4 days in 96-well plates.
6
Porcine Reproductive and Respiratory Syndrome Virus Protocols
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MARC-145, CRL-2843-CD163, and HEK-293T cell lines were kept in our laboratory (68 (link)). MARC-145 and HEK-293T cells were cultured in Dulbecco modified Eagle medium (DMEM; catalog no. 12100; Solarbio, Beijing, China) supplemented with 10% heat-inactivated fetal bovine serum (FBS; catalog no. 10270-106; Gibco, Waltham, MA) and antibiotics (100 U/mL penicillin, 100 μg/mL streptomycin; catalog no. P1400; Solarbio) in a humidified 37°C, 5% CO2 incubator. CRL-2843-CD163 cells were routinely maintained in Roswell Park Memorial Institute 1640 medium (RPMI 1640; catalog no. 31800; Solarbio), supplemented with 10% FBS and antibiotics at 37°C in a 5% CO2 incubator.
PRRSV strain HN07-1 (GenBank accession no.KX766378.1 ) and NADC30-like strain HNhx (GenBank KX766379 ) were previously isolated by our laboratory (69 (link), 70 (link)). PRRSV strain BJ-4 (GenBank AF331831 ) was kindly provided by Hanchun Yang from China Agricultural University. The infectious clone of HN07-1 (HN07-GFP) was constructed by inserting EGFP between PRRSV open reading frame 1b (ORF1b) and ORF2a through the reverse-genetics technique. The PRRSV strain used in the experiments was HN07-1 unless otherwise stated.
PRRSV strain HN07-1 (GenBank accession no.
7
Culturing OP9 and OP9-DL1 Stromal Cells
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OP9 and OP9-DL1 stromal cells were cultured in RPMI 1640 (Solarbio, Cat#31800) containing 2.05 mM L-glutamine, 23.8 mM sodium bicarbonate, 25 mM N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid (HEPES), 0.188 mM penicillin G sodium salt, and 0.0686 mM streptomycin sulfate, supplemented with 10% fetal bovine serum (FBS). For coculture experiments, OP9 or OP9-DL1 stromal cells were preseeded in a 96-well plate at a density of 1,000 cells/well 1 day earlier. Sorted progenitors were plated on the stromal cells and cultured with RPMI 1640 or Opti-MEM (GibcoTM, Cat#31985070) with 10% FBS, 50 μg/ml gentamycin sulfate (Sigma-Aldrich, Cat#345814), and 55 μM 2-mercaptoethanol (Sigma-Aldrich, Cat#M7522). SCF (20 ng/ml, Novus, Cat#NBP2-35150), Flt3L (20 ng/ml, Novus, Cat#427-FL-005), IL-7 (20 ng/ml, Novus, Cat#NBP2-35136), IL-33 (20 ng/ml, Novus, Cat#NBP2-35124), or IL-18 (20 ng/ml, Sino Biological, Cat#50073-MNCE) was added to the medium when specified. Half of the medium was removed and was replaced by fresh medium every 4–5 days. All cells were cultured at 37°C and 5% CO2.
8
Cell Culture Protocols for NSCLC Research
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The human NSCLC cell lines (A549, PC-9, and PC-9/GR) and other cell lines (16HBE and LO2) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). A549 and LO2 cells were cultured in RPMI-1640 (Solarbio, Beijing, China) containing 10% fetal bovine serum (FBS, Gibco, Carlsbad, CA, USA), and PC-9 and 16HBE cells were cultured in DMEM (Solarbio) with 10% FBS. The PC-9/GR cells, a gefitinib-resistant strain of human lung adenocarcinoma cell, were grown in DMEM with 10% FBS and gefitinib (800 ng/ml). All cell lines were cultivated at 37°C under 5% CO2.
9
Cholesterol Depletion and ER Stress in Breast Cancer
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MDA-MB-231 and BT-549 cells were obtained from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). The cells were maintained in a basal medium (RPMI-1640, Solarbio Science & Technology Co., Ltd., Beijing, China) containing 10% foetal bovine serum (FBS). To deplete cholesterol, cells were treated with HP-β-CD (BBI life Sciences, Shanghai, China) for 48 h. The regulation of cholesterol efflux and synthesis was carried out by culturing cells in an experimental medium containing LXR-623 (agonist of LXRα, cat. no. HY-10629; MedChemExpress, Monmouth Junction, NJ, USA), simvastatin, or DMSO (<0.1%) for 24 h. To induce ER stress, cells were incubated with tunicamycin (APExBIO technology, Houston, TX, USA, cat. no. B7417) for 24 h.
10
Cytotoxicity and Inflammatory Response of MP in A549 Cells
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A549 cells were inoculated in a 96-well plate with 1*104 cells per well and incubated for 24 h. The medium was replaced with fresh medium containing MP (MP: A549 = 1:5) and further incubated for 24 h with six replicates of each strain. Then the volumes were changed with RPMI 1640 containing CCK8 detection reagent (BBI, China) and incubated at 37°C for 2 h. The absorbance values (OD450) of each well were measured by a microplate reader.
Six-well plates were inoculated with 5*105 A549 cells per well and incubated for 24 h. The medium was replaced with fresh medium containing MP (MP: A549 = 1:5) and incubated for another 24 h. After incubation, the medium and cells were collected separately. The cells were resuspended with RPMI 1640 and used for detection of IL-1β (Solarbio, China), the medium was used for detection of IL-8 (USCN, China). A replicate experiment was conducted and the cells were collected for detection of caspase-3 activity (Solarbio, China). All the samples were detected by three replicate wells in each reagent kit.
Six-well plates were inoculated with 5*105 A549 cells per well and incubated for 24 h. The medium was replaced with fresh medium containing MP (MP: A549 = 1:5) and incubated for another 24 h. After incubation, the medium and cells were collected separately. The cells were resuspended with RPMI 1640 and used for detection of IL-1β (Solarbio, China), the medium was used for detection of IL-8 (USCN, China). A replicate experiment was conducted and the cells were collected for detection of caspase-3 activity (Solarbio, China). All the samples were detected by three replicate wells in each reagent kit.
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