L. mexicana (MNYC/BZ/1962/M379) promastigotes were cultured in M199 medium (Sigma-Aldrich, St. Louis, USA), supplemented with 2 µg/ml biopterin (Sigma-Aldrich), 2 µg/ml Hemin (Jena Bioscience GmbH, Jena, Germany), 25 mM HEPES and 50 units/ml of penicillin/streptomycin (Life Technologies/Thermo Fisher Scientific, Carlsbad, USA), and 10% heat-inactivated fetal bovine serum (FBS, BioSera Europe, Nuaillé, France). Metacyclic promastigotes and amastigotes were differentiated as previously described [44 (link)]. Prior to in vivo analysis, Leishmania cell lines were passaged through mice. Freshly isolated promastigotes were cultured in M199 medium (Sigma-Aldrich) containing 10% heat-inactivated FBS (Life Technologies) supplemented with 1% Basal Medium Eagle vitamins (Sigma-Aldrich), 2% sterile human urine, and 250 μg/ml amikacin (Bristol-Myers Squibb, New York, USA). For mice infections, the pH of the cultivation medium was adjusted to 5.5.
M199 medium
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom, Japan, Israel
M199 medium is a cell culture medium developed for the maintenance and growth of a variety of cell types. It provides a balanced formulation of essential nutrients, vitamins, and other components required for cell proliferation and survival in vitro.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (28 mentions)
Penicillin, by Merck Group (18 mentions)
Streptomycin, by Merck Group (18 mentions)
Rpmi 1640 medium, by Merck Group (11 mentions)
Heparin, by Merck Group (11 mentions)
L glutamine, by Merck Group (10 mentions)
Hemin, by Merck Group (10 mentions)
Hepes, by Merck Group (9 mentions)
Fetal bovine serum (fbs), by Merck Group (9 mentions)
Penicillin, by Thermo Fisher Scientific (9 mentions)
Streptomycin, by Thermo Fisher Scientific (9 mentions)
Sodium dodecyl sulfate (sds), by Merck Group (8 mentions)
Methylcellulose, by Merck Group (7 mentions)
Hepes, by Thermo Fisher Scientific (7 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (7 mentions)
Collagen 1, by Corning (6 mentions)
Biopterin, by Merck Group (6 mentions)
Penicillin streptomycin, by Merck Group (6 mentions)
Trypsin, by Merck Group (6 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (6 mentions)
207 protocols using m199 medium
1
Culturing L. mexicana Promastigotes and Differentiating Metacyclic and Amastigote Forms
2
hCG Bioactivity Assay in Leydig Cells
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The bioactive levels of circulating hCG were determined by the mouse testicular interstitial cell in vitro bioassay as described previously (Ding & Huhtaniemi 1989 (link), Rulli et al. 2002) . Briefly, decapsulated testes from adult WT males were dispersed with collagenase type I (0.15 mg/mL) in M199 medium (Sigma-Aldrich) for 5 min at 34°C. The supernatant was filtered through nylon mesh (mesh size 100 μm) and the cell suspension was washed twice with M199 medium supplemented with 0.1% (w/v) bovine serum albumin (BSA; Sigma-Aldrich) and 20 mM HEPES (Sigma-Aldrich). Testicular interstitial cells obtained using this technique are predominantly Leydig cells, as described previously (Ding & Huhtaniemi 1989) (link). Cells (50,000 cells/tube) were incubated with increasing concentrations of recombinant hCG as standard (AFP8456A, 20,000 IU/mg; NHPP, NIDDK), or with the serum samples, in a 95%O 2 /5%CO 2 atmosphere at 34°C for 4 h. After incubation, supernatants were recovered by centrifugation and frozen at -20°C. The testosterone concentration in the supernatants was measured by radioimmunoassay, according to a method described previously (Ratner et al. 2012) . The intra-and interassay coefficients of variation values were less than 12%.
3
Optimized protocol for chicken egg assessment
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Free-range, brown, chicken eggs (British Blacktail, gallus galuus) were purchased from a local supermarket (Waitrose, London, UK). Acetic acid, ≥99% was purchased from Fisher Scientific (Loughborough, Leicester, UK). EthylenediaminetetraAcetic acid (EDTA), hexamethyldisilane, ≥99%, (HMDS), cacodylate buffer, 0.1 M (CAB), 0.25% (v/v) trypsin-EDTA, 100 mM L-glutamine, Antibiotic-Antimycotic (AbAm), M199 medium, Giemsa stain and May-Grunwald stain were obtained from Merck (Poole, Dorset, UK). Glutaraldehyde was supplied by Agar Scientific (Stansted, Essex, UK). Epilife Medium with calcium and Human Keratinocyte Growth Supplement (HKGS) were purchased from Invitrogen Life Technologies (ThermoFisher, Leicester, UK). CellTiter 96® AQueous One Solution Cell Proliferation assay (i.e. MTS assay) and the CytoTox-ONE™ Homogeneous Membrane Integrity assay kits (i.e. LDH assay) were obtained from Promega (Southampton, Hampshire, UK). All other reagents and chemicals were obtained from Merck (Poole, Dorset, UK) unless otherwise stated.
4
HRMECs Glucose Metabolism Regulation
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The HRMECs were purchased from Cell Systems, and were routinely cultured in M199 medium (EMD Millipore) supplemented with 100 units of penicillin and 100 µg of streptomycin (both from Sigma-Aldrich; Merck KGaA) per milliliter of medium. All cells (passages 5-12) were cultured in grade plastic-ware and maintained in an atmosphere of 5% CO2 at 37°C.
For the establishment of the HG cell models, the HRMECs were cultured in conditioned medium with 5 mM [serving as the normal glucose (NG) group] or 30 mM (the HG group) D-glucose (Sigma-Aldrich; Merck KGaA) and incubated at 37°C with 5% CO2; the HG group was then treated with or without 1 µM of the NLRP3 inhibitor, CY-09, 1 µM of the ASK1 inhibitor, GS-444217 or 10 µM of the p38 inhibitor, SB203580 (all from MedChemExpress) for 24 h, respectively. Each inhibitor was dissolved in dimethyl sulfoxide (DMSO) to a concentration of 50 mM for use as stock solutions that were diluted to the required concentrations for in vitro experiments.
For the establishment of the HG cell models, the HRMECs were cultured in conditioned medium with 5 mM [serving as the normal glucose (NG) group] or 30 mM (the HG group) D-glucose (Sigma-Aldrich; Merck KGaA) and incubated at 37°C with 5% CO2; the HG group was then treated with or without 1 µM of the NLRP3 inhibitor, CY-09, 1 µM of the ASK1 inhibitor, GS-444217 or 10 µM of the p38 inhibitor, SB203580 (all from MedChemExpress) for 24 h, respectively. Each inhibitor was dissolved in dimethyl sulfoxide (DMSO) to a concentration of 50 mM for use as stock solutions that were diluted to the required concentrations for in vitro experiments.
5
Collagen-Methylcellulose Organoids for Cancer Research
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The basis of the collagen gels used was collagen type 1 (3 mg/mL, Corning) containing 10% 10× M199 medium (Sigma-Aldrich) and 0.22 M NaOH mixed with methylcellulose (Sigma-Aldrich) (MC) media (70% methylcellulose (1.5% w/v in DMEM and 2 mM I-glutamine), 20% DMEM and 10% FCS). 5*105 fibroblasts were suspended in 500 µL consisting of equal amounts of collagen suspension and MC media and poured into 24-well plates. Depending on the experimental setup, rSPARC was added to the gels as well. Empty collagen gels were casted as controls. Collagen gels were allowed to solidify in a humidified chamber at 37 °C for 30 min and then supplied with 1 mL DMEM containing 10% FCS. After 4 days, 5 × 105 cancer cells were added on top of both fibroblast containing as well as empty gels and allowed to attach for 24 h. Afterwards, gels were washed, supplied with DMEM containing 10% FCS and incubated at 37 °C in a humidified atmosphere with 7.5% CO2, renewing the media every 48 h. After additional 6 days, gels were washed with phosphate buffered saline (PBS), fixed with 1mL 4% formaldehyde (Histofix, PanReac AppliChem, Darmstadt, Germany) for 24 h and then processed for IHC.
6
3D Collagen Gel Contraction Assay
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3D‐collagen I gel contraction assay has been done as described earlier (Akcora et al, 2018 (link)). Briefly, a 5.0 ml collagen suspension with 3.0 ml collagen G1 (5 mg/ml, Matrix biosciences, Morlenbach, Germany), 0.5 ml 10× M199 medium (Sigma), 85 μl 1N NaOH (Sigma), and 1.415 ml of sterile water was prepared. This suspension was mixed with 1 ml (2 × 106) LX2 cells. 0.6 ml of the collagen‐gel suspension was plated in a 24‐well plates and incubated at 37°C/5% CO2 for 1 h. Polymerized gels were then incubated with serum‐free medium with or without TGFβ (5 ng/ml) together with 1 μM of PF8380 or Cpd17. Thereupon, gels were detached from the bottom of the culture wells. Pictures were taken with a digital camera at 0 h and 24 h. The area of the gel was digitally measured and normalized to the area of the inner size of the well in each image.
7
Collagen Gel Culture of Human Pluripotent Stem Cells
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A collagen suspension (5 ml) containing 3.0 ml collagen G1 (5 mg/ml, Matrix biosciences, Morlenbach, Germany), 0.5 ml 10x M199 medium (Sigma), 85 ul 1N NaOH (Sigma) and sterile water was mixed with 1.0 ml (2 × 106 cells) hPSCs. Collagen gel-cells suspension (0.6 ml/well) was plated in a 24-well culture plate and allowed to set for 1 h at 37 °C. Once set, gels were detached from the culture wells and 1 ml of serum free medium was added with or without TGF-β (5 ng/ml). To study the effect of FGF2 and FGF2-SPIONs, 1 ml of serum free medium with TGFβ (5 ng/ml) and FGF2 or FGF2-SPIONs (equivalent to 250 ng/ml FGF2) was added to the detached gels. Representative images were made at 96 h using a normal digital camera. Measurement of collagen gel diameter was performed using ImageJ (NIH).
8
Collagen Gel Contraction Assay to Evaluate Inhibitory Effect of Avagacestat
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A collagen gel contraction assay was performed as described previously41 (link) with minor modifications. We used this assay to examine the inhibitory effect of Avagacestat on the contractile activity of LX2 cells. A collagen suspension (5 ml) containing 3.0 ml Collagen G1 (5 mg/ml, Matrix biosciences, Morlenbach, Germany), 0.5 ml 10x M199 medium (Sigma), 85 ul 1N NaOH (Sigma) and sterile water was mixed with 1.0 ml (2 × 106 cells) human LX2 cells. Collagen gel-cells suspension (0.6 ml/well) was plated in a 24‐well culture plate and allowed to polymerize for 1 h at 37 °C. Once polymerized, 1 ml of 0.5% serum containing medium was added with or without TGFβ (5 ng/ml) together with 10 μM Avagacestat or PBS followed by detachment of the gels from the culture wells. Digital images were made at 0, 24, 48 and 72 h using a Nikon Coolpix digital camera (Nikon, Mississauga, ON, Canada). Measurement of collagen gel diameter at the indicated time points was performed using Image J imaging software (NIH, Bethesda, MD). Gels were measured by tracing around the edges of the gel disk and normalized with their respective well size in each image. Gel contraction experiments were performed in duplicates in three independent experiments.
9
Granulosa Cell Isolation from Mouse Ovaries
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Granulosa cell isolation was performed according to a method described in a previous study [20 (link)]. Ovaries from 3-week-old
wt mice were removed and dissected free of connective tissue. The ovaries were incubated in M199 medium (Sigma-Aldrich, St. Louis, MO, USA) containing 25 mM
HEPES and 0.1% BSA, and were punctured with a 27-gauge needle. Mixtures of granulosa cells and oocytes were filtered through cell strainers (40-µm nylon mesh,
BD Falcon, Bedford, MA, USA) that allowed granulosa cells but not oocytes to pass through. After centrifugation (5 min at 500 × g 4°C) cells
were collected.
wt mice were removed and dissected free of connective tissue. The ovaries were incubated in M199 medium (Sigma-Aldrich, St. Louis, MO, USA) containing 25 mM
HEPES and 0.1% BSA, and were punctured with a 27-gauge needle. Mixtures of granulosa cells and oocytes were filtered through cell strainers (40-µm nylon mesh,
BD Falcon, Bedford, MA, USA) that allowed granulosa cells but not oocytes to pass through. After centrifugation (5 min at 500 × g 4°C) cells
were collected.
10
Isolation and Culture of Rat Cardiomyocytes
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