Dulbecco's modified Eagle medium (DMEM), MEM non-essential amino acids and 0.25% trypsin/EDTA solution were purchased from Invitrogen (Gaithersburg, MD). Platelet-derived growth factor BB isotype (PDGF), GM6001 (also known as galardin or ilomastat) were obtained from Millipore (Billerica, MA). BB-94 (also known as batimastat) was purchased from Tocris (Bristol, UK). Fetal bovine serum (FBS), fatty acid-free and fraction V bovine serum albumin (BSA), RPMI vitamin mix, HEPES, DMSO, thymidine and Sodium bicarbonate were obtained from Sigma-Aldrich (St. Louis, MO). Penicillin, streptomycin, and amphotericin B were obtained from Lonza inc. (Walkersville, MD). Type I rat tail collagen was purchased from BD Biosciences (Bedford, MA). PureCol® Type I bovine collagen was purchased from Advanced BioMatrix, Inc. (San Diego, CA). Alexa Fluor 488 and Propidium Iodine (PI) were obtained from Molecular Probes, Inc. (Eugene, OR). Collagenase D from Clostridium histolyticum and RNase (DNase free) were purchased from Roche (Indianapolis, IN). Rabbit eyes were purchased from Pel Freez (Rogers, AR). Human glu-plasminogen was purchased from Haematologic Tech (Essex Junction, VT). Glass bottom dishes were purchased from MatTek (Ashland, MA). MT1-MMP mouse-anti-human monoclonal antibody (C9, Santa Cruz, TX)
Rpmi vitamin mix
Manufactured by Merck Group
Sourced in United States
RPMI vitamin mix is a laboratory reagent used as a component in cell culture media. It provides a standardized blend of essential vitamins to support the growth and maintenance of various cell lines in vitro. The specific composition and concentrations of the vitamins in the mix are formulated to optimize cellular metabolism and proliferation.
Automatically generated - may contain errors
Lab products found in correlation
Non essential amino acids (neaa), by Thermo Fisher Scientific (3 mentions)
Penicillin streptomycin amphotericin b, by Lonza (2 mentions)
Rat tail collagen type 1, by BD (1 mentions)
Trypsin edta solution, by Thermo Fisher Scientific (1 mentions)
Ilomastat, by Merck Group (1 mentions)
Bb 94, by Bio-Techne (1 mentions)
Propidium iodine, by Thermo Fisher Scientific (1 mentions)
Collagenase d from clostridium histolyticum, by Roche (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Hepes, by Merck Group (1 mentions)
Sodium bicarbonate, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Thymidine, by Merck Group (1 mentions)
Amphotericin b, by Lonza (1 mentions)
Penicillin, by Lonza (1 mentions)
Streptomycin, by Lonza (1 mentions)
Glass bottom dishes, by MatTek (1 mentions)
Ascorbic acid, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (1 mentions)
5 protocols using rpmi vitamin mix
1
Optimized Cell Culture Techniques for In Vitro Studies
2
Culturing Rabbit Corneal Keratocytes on Collagen
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To study cell behavior on the collagen substrates, primary rabbit corneal keratocytes (NRK) cells were extracted from New Zealand White Rabbit eyes (Pel-Freez, Rogers, AR, USA), as previously described [44 (link)]. Cells were cultured in basal media consisting of Dulbecco’s modified Eagle’s medium (DMEM; Sigma-Aldrich, St. Louis, MO, USA) supplemented with 1% RPMI vitamin mix (Sigma-Aldrich, St. Louis, MO, USA), 100 µM nonessential amino acids (Invitrogen), 100 µg/mL ascorbic acid, and 1% penicillin/streptomycin (Invitrogen) [45 (link),46 (link)]. First passage cells cultured for 4–5 days were used in all experiments.
3
Culturing Corneal Keratocytes from Rabbit Eyes
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Corneal keratocytes were isolated from rabbit eyes obtained from Pel Freez (Rogers, AR, USA) and cultured as previously described [5 (link)]. Cells were cultured in flasks with serum-free medium (basal medium) consisting of Dulbecco’s modified Eagle’s minimum essential medium with pyruvate (DMEM; Invtrogen, Carlsbad, CA, USA), supplemented with 1% RPMI vitamin mix (Sigma-Aldrich, St. Louis, MO, USA), 100 μM nonessential amino acids (Invitrogen, Carlsbad, CA, USA), 100 μg/mL ascorbic acid, and 1% penicillin/streptomycin/amphotericin B (Lonza Walkersville, Inc., Walkersville, MD, USA) to maintain the keratocyte phenotype [22 (link)]. For some experiments, previously published human corneal fibroblasts (HTK cells) were used [28 (link),29 (link)]. HTK cells were maintained in tissue culture flasks with DMEM containing 10% FBS and supplemented with 1% penicillin/ streptomycin/ amphotericin B, then serum-starved in basal media for 7 days prior to an experiment.
4
Corneal Keratocyte Isolation and Culture
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Corneal keratocytes were isolated from rabbit eyes obtained from Pel Freez (Rogers, AR) and cultured, as previously described [31 (link)]. Cells were cultured in flasks with a serum-free medium (basal medium) consisting of Dulbecco’s modified Eagle’s minimum essential medium with pyruvate (DMEM; Invtrogen, Carlsbad, CA) supplemented with 1% RPMI vitamin mix (Sigma-Aldrich, St. Louis, MO), 100 μM nonessential amino acids (Invitrogen), 100 μg/ml ascorbic acid, and 1% penicillin/streptomycin/amphotericin B (Lonza Walkersville, Inc., Walkersville, MD) to maintain the keratocyte phenotype [39 (link)].
5
Culturing and Differentiating Leishmania major
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Leishmania major Friedlin V1 strain (MHOM/IL/80/Friedlin; abbreviated as LmjF) parasites were grown at 26°C in M199 medium (US Biologicals) supplemented with 40 mM 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid (HEPES) pH 7.4, 50 μM adenosine, 1 μg ml−1 biotin, 5 μg ml−1 hemin, 2 μg ml−1 biopterin and 10% (v/v) heat-inactivated fetal calf serum [51 (link)], in some cases containing selective drugs. LmjF parasites expressing YFP (yellow fluorescent protein; SSU:IR1PHLEO-YFP) were described previously [51 (link)]. L. major LV39cl5 Δlpg1- [18 (link)] was cultured in the above media supplemented with 2 mM L-glutamine, 9 μg ml-1 folate and RPMI Vitamin Mix (Sigma). Infective metacyclic-stage parasites were recovered using the density gradient centrifugation method [36 (link)]. Prior to infection of host cells, purified metacyclic-stage parasites were opsonized with serum from C5-deficient mice as described [57 (link)]. For axenic amastigogenesis experiments shown in S3 Fig , day 3-stationary phase parasites were pelleted and resuspended in warm (37° C) RPMI 1640 media containing L-glutamine and then cultured for 24 h at 37° C in CO2 incubator.
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