Candida albicans (ATCC 90028, American Type Culture Collection, Manassas, VA, United States) was received from the Department of Microbiology, Institute of Medical Sciences, Banaras Hindu University, Varanasi, India. After collection, the culture was revived in YPD (Yeast extract peptone) broth (supplemented with 1% glucose) at neutral pH (7.2) and preserved in 25% glycerol at-80oC for further investigation. Polyethyleneimine, silver nitrate (AgNO3), calcofluor white, bovine serum albumin (BSA), tryptophan, tyrosine, amphotericin B, and other routine chemicals were obtained from Sigma Aldrich Chemicals Private Limited (Bangalore, Karnataka, India). RPMI (Rosewell Park Memorial Institute media) and other media constituents were obtained from Hi-Media Laboratories Ltd. (Mumbai, Maharashtra, India). Plasticware was obtained from Tarsons Products Limited (Kolkata, West Bengal, India). The solvents, including ultra-purified water, were obtained from Merck Life Science Private Limited (Bangalore, Karnataka, India). All of the reagents were of analytical grade. The generated experimental data were plotted and analyzed using Origin 8.5 software (Origin Lab Corporation, Northampton, MA, United States).
Roswell park memorial institute (rpmi)
Manufactured by HiMedia
Sourced in India
RPMI is a culture medium used in cell and tissue culture applications. It provides a balanced salt solution and nutrients necessary for the growth and maintenance of various cell types. RPMI is commonly used in research and laboratory settings.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by HiMedia (5 mentions)
Dulbecco modified eagle medium (dmem), by HiMedia (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Mcf 7, by HiMedia (2 mentions)
Huvecs, by Lonza (2 mentions)
Origin 8, by OriginLab (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Chitosan, by Merck Group (1 mentions)
Sodium chloride (nacl), by Thermo Fisher Scientific (1 mentions)
Acetone, by Thermo Fisher Scientific (1 mentions)
Calcium chloride, by Merck Group (1 mentions)
Phosphotungstic acid, by Merck Group (1 mentions)
Triethylamine, by Merck Group (1 mentions)
Ultrapure water, by Merck Group (1 mentions)
Sodium hydrogen carbonate, by Merck Group (1 mentions)
Iodomethane, by Merck Group (1 mentions)
Sabouraud dextrose agar media, by HiMedia (1 mentions)
Cholesterol, by HiMedia (1 mentions)
Dicetyl phosphate, by Merck Group (1 mentions)
N methylpyrrolidone, by Merck Group (1 mentions)
10 protocols using roswell park memorial institute (rpmi)
1
Candida albicans ATCC 90028 Cultivation
2
Cell Culture Protocols for Diverse Cell Lines
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HEK-293T (Human Embryonic Kidney 293T cells), MCF-7 (Human breast adenocarcinoma cell line, p53 Wt), H1299 (non-small cell lung carcinoma, p53 null), HeLa (Human cervical adenocarcinoma cell line) were maintained in Dulbecco's Modified Eagle's Medium (DMEM; Himedia Laboratories) supplemented with 10% fetal bovine serum (Gibco, Invitrogen), 100 units penicillin, 0.1 mg streptomycin and 0.25 µg amphotericin B per ml at 37°C in the presence of 5% CO2 in a humidified incubator. MOLT-3 (T-lymphoblastoid cell line, p53 Wt), MOLT-3 p53 knockdown cells (MOLT-3 p53 kd), Jurkat E6.1 (T-lymphocyte) were cultured in RPMI (Himedia Laboratories) supplemented with 10% fetal bovine serum (Gibco, Invitrogen), 100 units penicillin, 0.1 mg streptomycin and 0.25 µg amphotericin B per ml at 37°C in the presence of 5% CO2 in a humidified incubator. Transfections were carried out using Lipofectamine 2000 (Invitrogen, U.S.A.) and Polyethyleneimine, Linear (MW 25 000, Polysciences Inc., U.S.A.) reagents using the manufacturers’ protocols.
3
Culturing Pro-Monocytic and T-Cell Lines
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U1 (a pro-monocytic cell line) and J1.1 (a T cell line) cells were cultured in RPMI (Himedia Laboratories) with or without 10% fetal bovine serum (Gibco, Invitrogen), 100 units penicillin, 0.1 mg streptomycin and 0.25 μg amphotericin B per ml at 37 °C in presence of 5% CO2 in a humidified incubator. fetal bovine serum used during the experiments was not heat inactivated. This was done to avoid degradation of serum components. All experiments were performed on cell lines only.
4
Natamycin-Loaded Chitosan Nanoparticles
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Natamycin was procured from AKUMS Drugs & Pharmaceuticals Ltd. (Haridwar, UK, India). Dicetyl phosphate (DCP), chitosan (low molecular weight), triethylamine, iodomethane, and N-methylpyrrolidone (NMP) were purchased from Sigma-Aldrich, India. Sterile blank disks, MOPS (3-(N-morpholino)propanesulfonic acid) buffer, Sabouraud dextrose agar media, RPMI (Roswell Park Memorial Institute) media, and cholesterol (CHOL) were procured from HiMedia, Mumbai, India. Span 60 (Sp60) and sodium hydroxide were purchased from CDH (Central Drug House; New Delhi, India). Sodium chloride and acetone were purchased from Fisher Scientific India. Cellulose acetate membrane filter (syringe filter) was purchased from Chromatopak. Methanol and chloroform (Merck Life Science Pvt. Ltd., Mumbai, India), phosphotungstic acid (LobaChemie), sodium hydrogen carbonate, and calcium chloride (CDH, New Delhi) were obtained from the departmental chemical store. The marketed anti-keratitis eye drops, Nataforce (5% w/v, Mankind), was purchased from Netmeds. Ultrapure water (Millipore, Bedford, MA) was used throughout the studies. All other chemicals were of the highest grade commercially available.
5
MTT Assay: Evaluating Cell Viability
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Thymol and 4',6-diamidino-2-phenylindole (DAPI) staining dye were obtained from Sigma-Aldrich (St. Louis, MO, USA). MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide], Fetal bovine serum, RPMI (Roswell Park Memorial Institute)-1640 media, and antibiotics were procured from Himedia India, Ltd. (Mumbai, India). Caspase-3 assay kit was purchased from BioVision U.S.A. cDNA synthesis kit (Product name: Verso), and qPCR Kit (DyNAmoColorFlash SYBR Green) were procured from Thermo Scientific, USA.
6
Silver Nitrate Cell Culture Reagents
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AgNO3 was procured from Sigma Aldrich, USA. RPMI, Dulbecco's Modified Eagle Medium (DMEM), antibiotics, and fetal bovine serum were purchased from Himedia Ltd., India. All the other reagents of analytical grade were purchased from Loba Chemie, India.
7
Authenticated Cell Lines for Cancer Research
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The breast cancer cell lines, MCF7 (RRID:CVCL_0031) and MDA-MB-468 (RRID:CVCL_0419) were purchased from cell repository, NCCS (Pune, India) in 2016. At the time of procurement, cells were authenticated using 16 STR markers (AmpFISTR Identifier Plus PCR Amplification Kit, Applied Bio Systems) by the repository. Cell lines were found free from Mycoplasma contamination (MycoAlert Mycoplasma Assay Kit, Lonza) with the latest test done in the year 2018. During experiments, cells were passaged for two years. MCF7 and MDA-MB-468 were cultured in DMEM and RPMI (HiMedia), respectively, with 10% FBS (US origin, HiMedia), ampicillin (100 IU/mL, HiMedia), and streptomycin (100 mg/mL, HiMedia). HUVECs were purchased from Lonza and cultured in complete EGM-2 endothelial cell growth medium (Lonza). These cells were tested for Mycoplasma contamination by the supplier and used within three months of time for various experiments. All cells were maintained in a humidified chamber at 37 C temperature and 5% CO 2 .
8
Cell Line Preparation and Compound Screening
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Four different cell lines, namely, MCF7 (human breast cancer), HCT116 (human colon cancer), NCI-H460 (human non-small-cell lung cancer), and JURKAT (human T-cell lymphoma) cell lines, were obtained from NCCS, Pune, India. All the cell lines were cultured in DMEM (Dulbecco's modified Eagle's medium), except JURKAT, which was cultured in RPMI (Roswell Park Memorial Institute) media supplemented with 10% FBS (fetal bovine serum) (Himedia, India), containing penicillin (100 I.U/mL) and streptomycin (100 μg/mL) at 37°C in a humidified atmosphere of 5% CO2 in air. For compound testing, the adherent cells were trypsinized, counted, and seeded in 96-well plates for viability studies at a density of 20 × 103 cells per well, where they were left to adhere overnight before the experiments with our compounds. The compounds under assay were not added until the wells were observed to show at least 80% confluence [46 (link)].
9
Breast Cancer Cell Culture Protocol
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Human Breast cancer cell lines MCF7 and MDA‐MB‐231 were procured from the cell repository, National Center for Cell Science (Pune, India). MCF7 cells were grown in Dulbecco's Modified Eagle Media (DMEM; Himedia, Mumbai, India) supplemented with 1X Penicillium‐Streptomycin‐Amphotericin B (PSA; Himedia) and 10% fetal bovine serum (FBS; Himedia, US origin). MDA‐MB‐231 cells were grown in Roswell Park Memorial Institute Media (RPMI; Himedia) supplemented with 1× PSA (Himedia) and 15% FBS (Himedia, US origin). HUVECs were purchased from Lonza (Walkersville, MD, USA) and maintained in complete Endothelial Cell Growth Media‐2 (EGM‐2, Lonza, Walkersville, MD, USA). Cells were grown in the incubator (Eppendorf, Hamburg, Germany) at 37 °C temperature and 5% CO2.
10
3D Vascular Lung Model: Cell Culture Protocol
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For the 3D vascular lung model three types of cell were grown:
1.
A549 cells were grown at 37 o C in the growth medium DMEM (HIMEDIA #Cat
No-AT007) supplemented with 10% (v/v) fetal bovine serum under the atmosphere containing 5% CO2. Cells were subcultured after reaching 80-90% confluence.
2. HUVEC cells were grown at 37 o C in the Endothelial cell basal medium-2 (LONZA #Cat No-CC-3156 and CC-4176) under the atmosphere containing 5% CO2. Cells were subcultured after reaching 80-90% confluence.
3. HL60 cells were grown at 37 o C in the growth medium RPMI (HIMEDIA #Cat No-AT028) supplemented with 10% (v/v) fetal bovine serum under the atmosphere containing 5% CO2. Cells were subcultured after reaching 80-90% confluence.
1.
A549 cells were grown at 37 o C in the growth medium DMEM (HIMEDIA #Cat
No-AT007) supplemented with 10% (v/v) fetal bovine serum under the atmosphere containing 5% CO2. Cells were subcultured after reaching 80-90% confluence.
2. HUVEC cells were grown at 37 o C in the Endothelial cell basal medium-2 (LONZA #Cat No-CC-3156 and CC-4176) under the atmosphere containing 5% CO2. Cells were subcultured after reaching 80-90% confluence.
3. HL60 cells were grown at 37 o C in the growth medium RPMI (HIMEDIA #Cat No-AT028) supplemented with 10% (v/v) fetal bovine serum under the atmosphere containing 5% CO2. Cells were subcultured after reaching 80-90% confluence.
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