Betulinic acid (BA, CAS no. 472-15-1), 1, 1-diphenyl-2-picrylhydrazyl free radical (DPPH, CAS no. 1707-75-1), 2,4,6-Tris (2-pyridyl)-s-triazine (TPTZ, CAS no. 3682-35-7), triton X-100 (CAS no. 9002-93-1) and methyl methanesulfonate (MMS, CAS no. 66-27-3) were purchased from Sigma–Aldrich (St. Louis, MO, USA). Ethanol and aluminium chloride anhydrous (AlCl3, CAS no. 7446-70-0) were purchased from Merck Specialties’ (Mumbai, India). Acetonitrile (HPLC grade) and dimethylsulfoxide (DMSO) was procured from Qualigens (Mumbai, India). Other reagents like ethylene diamine tetra acetic acid (EDTA) di-sodium salt, normal melting point agarose (NMPA) and low melting point agarose (LMPA), HiSep™ LSM 1084, Tris buffer, phosphate-buffered saline (PBS) (Ca2+, Mg2+ free), RPMI-1640 media, were procured from Hi Media, Mumbai, India. Ascorbic acid, gallic acid, Folin–Ciocalteu reagents were purchased from SRL (Mumbai, India). Sodium di-hydrogen phosphate (NaH2PO4), di-sodium hydrogen phosphate (Na2HPO4), hydrogen peroxide (H2O2), ferric chloride (FeCl3), sodium hydroxide (NaOH) and sodium carbonate (Na2CO3) were obtained locally and were of analytical grade.
Rpmi 1640 media
Manufactured by HiMedia
Sourced in India, United States
RPMI-1640 media is a common cell culture medium used for the in vitro cultivation of various cell types, including human and animal cells. It provides essential nutrients and growth factors required for cell growth and maintenance.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (6 mentions)
Amino acids, by HiMedia (3 mentions)
Dmem media, by HiMedia (3 mentions)
Fetal bovine serum (fbs), by HiMedia (3 mentions)
Apoptosis detection kit, by Merck Group (2 mentions)
Coumaric acid, by Merck Group (2 mentions)
Fluorescein isothiocyanate fitc conjugated annexin 5, by Merck Group (2 mentions)
Rnase a, by Merck Group (2 mentions)
Luminol, by Merck Group (2 mentions)
Bradford reagent, by Merck Group (2 mentions)
Methylthiazolyldiphenyl tetrazolium bromide mtt, by Sisco Research Laboratories (2 mentions)
Antibiotic antimycotic solution, by HiMedia (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Antibiotic solution, by HiMedia (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Betulinic acid, by Merck Group (1 mentions)
Methyl methanesulfonate, by Merck Group (1 mentions)
Tris buffer, by HiMedia (1 mentions)
Phosphate buffered saline (pbs), by HiMedia (1 mentions)
19 protocols using rpmi 1640 media
1
Antioxidant and Genotoxicity Assays
2
IL-33 Stimulation of THP-1 Cells
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THP-1 cells (ATCC®) were maintained in RPMI 1640 media (HiMedia) supplemented with 10% (v/v) FBS and 1% (v/v) antibiotic- antimycotic solution (Thermo Fisher Scientific, Cat# 15240062) at a density of 2 × 105 cells/mL in a humidified incubator at 37 °C with 5.0% CO2 [16 (link)]. The cells were stimulated with 50 ng/mL human rIL-33 (R&D Systems) for different time intervals (i.e., 0, 5, 10, 15, 30, 40, 60, 120 and 240 min). The 50 ng/mL concentration was based on previous studies [17 (link),18 (link),19 (link),20 (link)]. All the treatments were performed in triplicate. The cell pellets were washed with 1% PBS and lysed with lysis buffer (4% sodium dodecyl sulfate (SDS) in 50 mM triethylammonium bicarbonate (TEABC), sodium orthovanadate (1 mM), sodium pyrophosphatase (2.5 mM), and beta-glycerophosphate (1 mM)) and heated at 95 °C on a dry bath for 10 min, followed by centrifugation at 12,000× g for 20 min. Proteins were extracted by probe sonication using Q-Sonica (Cole-Parmer, Mumbai, India) and the concentration was estimated using Bicinchoninic acid assay (BCA) (Pierce, Waltham, MA, USA).
3
Cell Viability and Apoptosis Assay
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RPMI-1640 media, DMEM media and other necessary chemicals like antibiotics, amino acids, etc. were purchased from HIMEDIA (Mumbai, India). Fetal bovine serum (FBS) was purchased from HyClone (Thermo Scientific Hy-Clone, Logan, Utah, USA). Methylthiazolyldiphenyl-tetrazolium bromide (MTT) was purchased from Sisco Research Laboratory (Mumbai, India). Cisplatin, ribonuclease, Fluorescein isothiocyanate (FITC) conjugated Annexin V, apoptosis detection kit, Z-vad FMK, RNaseA, Bradford reagent, luminol and coumaric acid were purchased from Sigma (Missouri, USA). Antibodies were purchased from abcam (Cambridge, UK) and Cell Signaling Technology (Danvers, MA, USA). Other essential chemicals used in this study were of the analytical grade.
4
Investigating Cellular Responses to Oxidative Stress
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HiEndoXL cell expansion medium (AL517), fetal bovine serum (FBS), EnVzyme, Bovine serum albumin (BSA), RPMI 1640 media, and antibiotic–antimycotic solution were purchased from Hi-media Laboratories (Mumbai, India). The iScript cDNA synthesis kit and SYBR Green Master mix were procured from Bio-Rad (Hercules, CA, USA). The miScript II RT kit and miRNeasy mini kit were purchased from Qiagen (Hilden, Germany). Antibodies SIRT-1 (SC-74465) and secondary anti-mouse (sc-516102) were procured from Santacruz biotechnology Inc. PGC-1α (ab54481), RNA-later stabilizing solution were purchased from Ambion Inc. (Naugatuck, CT, USA). β-actin (PA1-183), secondary anti-rabbit (32460), ox-LDL (L34357), and TRIzol were procured from Invitrogen (Carlsbad, CA, USA). CORM-A1, hematoxylin, eosin, Direct Red 80, and phorbol 12-myristate 13-acetate (PMA) were purchased from Sigma Aldrich (St. Louis, MO, USA). Methanol, dimethyl sulphoxide (DMSO), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) were purchased from Sisco Research Laboratory pvt. Ltd. (Mumbai, India).
5
Liver Leukocyte Isolation Protocol
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Liver-infiltrating leukocytes were isolated by mechanical disruption of liver biopsy in Medimachine for 1 min (BD Biosciences, San Jose, CA, USA) according to the technical instructions of the manufacturer. Cells were collected in complete RPMI 1640 media (10% fetal bovine serum, 2 gm/l sodium bicarbonate, and 1% penicillin/streptomycin) (Himedia, USA), and further experiments were performed.
6
Isolation of Human Neutrophils
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Neutrophils were isolated using an EasySep Direct Human Neutrophil isolation kit (StemCell Technologies, Vancouver, BC, Canada) according to the manufacturer’s protocol. Briefly, blood was withdrawn from healthy donors with prior consent in EDTA vials and the antibody cocktail was added to it (50 μL/mL of blood) for 5 min. After that, rapidsphere beads (50 μL/mL of blood) were added and incubated for 5 min. The suspension was diluted with Ca2+/Mg2+ free PBS and kept in the magnetic stand for 5 min. The suspension was carefully decanted in another tube in one continuous flow. The same amount of rapidsphere beads was added to the suspension for 5 min and then kept on the magnetic stand again for 5 min. This step was repeated again but without the addition of rapidsphere beads until a clear suspension was obtained. Finally, the pure neutrophil suspension was centrifuged at 800 rpm for 5 min at RT. The cells were then resuspended into RPMI-1640 media (HiMedia, Mumbai, India) and were counted using a hemocytometer before downstream applications.
7
Maintenance and Subculture of Cell Lines
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MhCT12-E, MhCT12-F, MhCT08-F, MhCT08-E43 (link),44 (link) MhCL03-F, MhCB05-F and MhCA04-F44 (link) were maintained RPMI- 1640 media (#AT222A; HiMedia) with 20% Fetal Bovine Serum (FBS; 10,270–106; Gibco), 1X penicillin- streptomycin (#15,140,122; Gibco) and subcultured every 48 h.
8
Synthesis and Characterization of Curcumin-Functionalized Nanoparticles
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Magnesium(II) acetate tetrahydrate (99%), zinc(II) acetate dihydrate (99.5%), N-hydroxyl succinimide (97%), 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride (EDC∙HCl) extra pure (99%), 3-carboxybenzeneboronic acid (97%), potassium hydroxide pellets, disodium dihydrogen phosphate dihydrate, potassium dihydrogen phosphate, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and sodium chloride were purchased from Sisco Research Laboratory (Mumbai, India). 3-aminopropyl triethoxysilane (APTES) with a purity of 99% was purchased from Sigma-Aldrich (St. Louis, Missouri, USA). Ethyl alcohol (EtOH) and dimethyl sulfoxide (DMSO) were purchased from Merck (Kenilworth, New Jersey, United States). Curcumin, DMEM media, RPMI-1640 media, amino acids and antibiotics were purchased from Hi-Media (Mumbai, India). Fetal bovine serum (FBS) was purchased from Thermo Scientific Hy-Clone (Logan, Utah, USA).
9
Cytotoxicity Assay on H-PBMC Cells
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The cytotoxicity assay was performed on Human Peripheral Blood Mononuclear cells (H-PBMC, Himedia, India) according to the manufacturer’s instructions. The H-PBMC (40,000 cells/well in 100 μL volume) suspension in RPMI-1640 media (containing 10% fetal bovine serum and 50 U/mL antibiotic-antimycotic solution) (Himedia, India) was dispensed into 96-well polystyrene tissue culture-treated flat-bottom plates (Corning, U.S.). The plates were incubated for 48 h in a CO2 (5%) incubator at 37°C. After 48 h, the compounds (Ar1-Ar24) were added to the plates in a fresh medium and again incubated for 24 h in a CO2 (5%) incubator at 37°C. Next, MTT (1 mg/mL) was added to the wells, followed by incubation for 4–5 h. The formazan crystals were solubilized using a solubilizing solution (40% (v/v) dimethyl formamide in 2% (v/v) glacial acetic acid added with 16% sodium dodecyl sulfate), and the absorbance (OD570nm) was measured.
10
Immune cell assay reagents
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RPMI 1640 media, Fetal Bovine Serum (FBS) and l -glutamine, phosphate buffer saline (PBS) were purchased from HiMedia (Mumbai, India). Penicillin and streptomycin were obtained from Life Technologies, USA. Antibodies against IL-2 (ab180780), IL-6 (ab208113), anti-mouse HRP linked secondary antibody, anti-rabbit HRP linked secondary antibody were purchased from Abcam (MA, USA). Antibodies against TGF-β, Actin were purchased from Santa Cruz Biotechnology (Dallas, Texas USA). Anti-CD4, anti-CD8, anti-CD19, anti CD3e antibody (FITC conjugated), anti Ki67 antibody (PE conjugated), anti-IL-2, and IL-6 antibodies (PE conjugated) were purchased from BD Biosciences, USA. PMA, Ionomycin, Brefeldin A, saponin were purchased from Sigma Aldrich, India. Vectashield mounting medium was from Vector Laboratories, Inc. (CA, USA). MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide), Bovine serum albumin (BSA) were purchased from SRL India. SRBC was procured from Indian Veterinary Research Institute, Eastern Regional Station, Kolkata. Cyclophosphamide was purchased from Abcam.
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