Chick limb bud cells were plated for triplicate in 48 well plates at a density of 3 × 105 cells/10 uL in the DMEM-HG medium (Life Technologies) according to Ahrens et al. (1977) (link) and supplemented with 10% of FBS (Life Technologies), 100 U/mL penicillin and 100 mg/mL streptomycin (Sigma-Aldrich), 1× non-essential amino acids (Life Technologies), and 1× GlutaMAX (Life Technologies) in a humidified incubator containing 5% CO2 at 37°C for 2 h. Posteriorly, micromass were flooded in DMEM-HG medium without serum. After 72 h of incubation, the micromass were fixed in Khale’s fixative for Alcian blue staining or in 4% PFA for in situ hybridization. All micromass assays were performed by triplicate.
Dmem hg medium
Manufactured by Thermo Fisher Scientific
Sourced in United States
DMEM-HG medium is a high-glucose Dulbecco's Modified Eagle's Medium, a widely used cell culture medium. It supports the growth and maintenance of a variety of cell types.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (7 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (3 mentions)
Glutamax, by Thermo Fisher Scientific (3 mentions)
Sant 1, by Merck Group (2 mentions)
Noggin, by R&D Systems (2 mentions)
Retinoic acid, by Merck Group (2 mentions)
Mineral oil, by Merck Group (1 mentions)
Polydimethylsiloxane pdms prepolymer, by Dow (1 mentions)
Sephadex g50 columns, by GE Healthcare (1 mentions)
Sylgard 184, by Dow (1 mentions)
Anhydrous calcium chloride, by Sinopharm (1 mentions)
Delta t dishes, by Bioptechs (1 mentions)
Cos 7 cells, by American Type Culture Collection (1 mentions)
Calyculin a, by Merck Group (1 mentions)
Glutamax, by American Type Culture Collection (1 mentions)
X tremegene hp dna transfection reagent, by Roche (1 mentions)
X tremegene hp, by Roche (1 mentions)
Gentamicin, by Thermo Fisher Scientific (1 mentions)
Proline, by Merck Group (1 mentions)
Fungizone, by Thermo Fisher Scientific (1 mentions)
16 protocols using dmem hg medium
1
Chick Limb Bud Cell Micromass Culture
2
Osteogenic and Chondrogenic Differentiation of Murine Limb Cells
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For osteogenic differentiation, mouse limb subpopulations and total cells were directly sorted at 30,000 cells/cm2 in 96 well plates to reach the 80% confluence. Cells for each condition were incubated for 15 days in 1 mL of Complete MesenCult Osteogenic Medium (StemCell Technologies, Vancouver, BC, Canada). Debris or detached cells were washed with PBS, and the medium was replaced with fresh medium every 3–4 days. After 15 days of induction, differentiated cells were washed with PBS and fixed in 4% paraformaldehyde for 30 min at 4°C. For alizarin red staining, cells were fixed in 10% of formaldehyde-PBS for 10 min at room temperature. After washing, cells were stained with 0.2% Alizarin S-Red solution (pH 4.2) for 20 min. Excess of alizarin staining was washed twice with water for further image acquisition.
Chondrogenic differentiation was evaluated by micromass assays. Freshly isolated subpopulations and total cells were directly seeded in 48 well plates at 3 × 105 cells in 10 μL of DMEM-HG medium (Life Technologies), supplemented with 10% FBS (Life Technologies). After permit cell attachment for 2 h, micromass was flooded in DMEM-HG medium (Life Technologies). Cultures were maintained for 3 days under a 5% CO2 atmosphere until Alcian blue staining. Chondrogenic and osteogenic differentiation images were acquired with an AxioZoom V.16 fluorescence microscope (Carl Zeiss).
Chondrogenic differentiation was evaluated by micromass assays. Freshly isolated subpopulations and total cells were directly seeded in 48 well plates at 3 × 105 cells in 10 μL of DMEM-HG medium (Life Technologies), supplemented with 10% FBS (Life Technologies). After permit cell attachment for 2 h, micromass was flooded in DMEM-HG medium (Life Technologies). Cultures were maintained for 3 days under a 5% CO2 atmosphere until Alcian blue staining. Chondrogenic and osteogenic differentiation images were acquired with an AxioZoom V.16 fluorescence microscope (Carl Zeiss).
3
Chick Hindlimb Bud Dissociation
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Fertilized White Leghorn chicken eggs (ALPES, Puebla, Mexico) were incubated at 38°C and staged according to Hamburger and Hamilton (1951) (link). The eggs were windowed at stage 22–23HH; embryos were removed from the egg and washed in PBS 1×. Whole hindlimb buds were dissected out and dissociated with 2 mg/mL collagenase type IV (Life Technologies) in Hanks Solution at 37°C for 5 min. Limb buds were resuspended in DMEM-HG medium supplemented with 10% of FBS (Life Technologies) to inactivate collagenase and pipetted until a single-cell suspension was obtained. The cell suspension was centrifuged at 1,100 rpm for 5 min. After obtention, cells were resuspended in DMEM-HG medium (Life Technologies).
4
Microfluidic Fabrication of Liposomes
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Sodium
alginate (PRONOVA UP LVM) was purchased
from NovaMatrix (Norway). All lipids were purchased from Avanti Polar
Lipids, Inc. (Alabaster, AL, USA). Ethylenediamine tetra-acetic acid
disodium salt dihydrate (Na2EDTA) and calcium chloride
anhydrous were purchased from Sinopharm Chemical Reagent Co., Ltd.
(Shanghai, China). Sodium chloride was purchased from Dieckmann (China).
Mineral oil and trichloro(1H,1H,2H,2H-perfluoro-octyl) silane were
purchased from Sigma (Sigma-Aldrich, Milwaukee, WI). Negative photoresist
SU-8 2050 and SU8 developer were obtained from Chestech (Rugby, UK).
Polydimethylsiloxane (PDMS) prepolymer and the curing agent (Sylgard
184) were obtained from Dow Corning (Midland, MI, USA). Sephadex G50
columns were purchased from GE Healthcare Life Sciences (Pittsburgh,
PA). Dulbecco’s Minimum Essential Medium (DMEM, GlutaMAXTM-1)
and DMEM-HG medium were purchased from Gibco, Invitrogen.
alginate (PRONOVA UP LVM) was purchased
from NovaMatrix (Norway). All lipids were purchased from Avanti Polar
Lipids, Inc. (Alabaster, AL, USA). Ethylenediamine tetra-acetic acid
disodium salt dihydrate (Na2EDTA) and calcium chloride
anhydrous were purchased from Sinopharm Chemical Reagent Co., Ltd.
(Shanghai, China). Sodium chloride was purchased from Dieckmann (China).
Mineral oil and trichloro(1H,1H,2H,2H-perfluoro-octyl) silane were
purchased from Sigma (Sigma-Aldrich, Milwaukee, WI). Negative photoresist
SU-8 2050 and SU8 developer were obtained from Chestech (Rugby, UK).
Polydimethylsiloxane (PDMS) prepolymer and the curing agent (Sylgard
184) were obtained from Dow Corning (Midland, MI, USA). Sephadex G50
columns were purchased from GE Healthcare Life Sciences (Pittsburgh,
PA). Dulbecco’s Minimum Essential Medium (DMEM, GlutaMAXTM-1)
and DMEM-HG medium were purchased from Gibco, Invitrogen.
5
COS-7 Cell Culture and Transfection
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Cell culture was performed as reported previously23 (link) and reiterated here. COS-7 cells were obtained from ATCC (Manassas, VA, USA, catalog number CRL-1651) and maintained in standard DMEM-HG medium supplemented with 10% (vol/vol) heat-inactivated FBS, 1 mM sodium pyruvate and 2 mM Glutamax in a 5% CO2 incubator at 37 °C. For imaging, the cells were plated in Bioptechs Delta-T dishes (Bioptechs, Butler, PA, USA, product number 04200417B) and grown to ~ 70% confluency. Transient transfections were done with various plasmids according to manufacturer’s instructions using 1–2 μg DNA and 3ul of X-tremeGene HP DNA transfection reagent (Roche, Manheim, Germany, product number 06366236001). The transfected cells were incubated at 37 °C and 5% CO2 incubator for 24–48 h before imaging. The cell culture reagents DMEM-HG medium (catalog number 11960), fetal bovine serum (FBS, catalog number 10082), sodium pyruvate (catalog number 11360) and Glutamax (catalog number 35050) were obtained from Invitrogen (Life Technologies, Grand Island, NY, USA). Calyculin A was obtained from Sigma (catalog number C 5552).
6
Cultivating and Transfecting COS 7 Cells
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COS 7 cells (product no. CRL-1651; ATCC) were cultivated at 37 °C under 5% CO2 in Bioptechs Delta-T dishes (product no. 04200417B; Bioptechs) and grown in standard DMEM-HG medium (product no. 11960; Invitrogen, Life Technologies) with 2 mM Glutamax (product no. 35050; Invitrogen), 1 mM sodium pyruvate (product no. 11360; Invitrogen), and 10% (vol/vol) heat-inactivated FBS (product no. 10082; Invitrogen). Transfections were performed using XtremeGene HP (product no. 06366236001; Roche) and incubated 24–48 h before imaging.
7
EAEC042 Infection of Intestinal Colonoids
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EAEC042 strain was isolated from a child with diarrhea in Lima, Peru, and was shown to cause diarrhea in adult volunteers60 (link). E. coli HS is a human commensal isolate that was originally isolated at the Walter Reed Army Institute of Research61 (link). Isogenic 042pic and 042pic complemented with Pic in-trans were generated in previous studies27 (link). All strains are available from our strain collection. Strains were grown in L-broth supplemented with appropriate antibiotics. All antibiotics were purchased from Sigma Chemical Co. (St. Louis, MO). For colonoid infections, strains were grown from frozen stocks (−80 °C) at 37 °C on Luria broth (LB) agar plates (Difco) 2- days prior to experiments. The day before infections, single colonies were inoculated in 5 ml of L-broth and grown overnight with vigorous shaking at 37 °C. The day of the infections, overnight cultures were diluted 1:50 (V/V) with fresh DMEM-HG medium (Invitrogen, USA) to induce the master regulator of virulence AggR in EAEC042. Subcultures were grown at 37 °C with shaking to the mid-log phase (OD600 = 0.6). Subsequently, bacteria were adjusted to 108 CFU/ml in sterile PBS, and 10 μL (1 × 106) was added to the apical surface of colonoid monolayers. E. coli infections were allowed to progress for 6 h.
8
Senescent MSC Chondrogenesis Assay
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Senescence was induced in the cells by 20 Gy ionizing radiation using an RS320 X-Ray machine (X-Strahl, Camberley, United Kingdom) (Voskamp et al., 2021 (link)). MSCs in a monolayer were irradiated in a T175 flask (60%–70% confluency) for 22 min (20 Gy). Then, 24 h post-irradiation, the cells were trypsinized and seeded at a 9,600 cell/cm2 density. Mock-irradiated MSCs were used as non-senescent controls and seeded at 2,300 cells/cm2. Seven days post-irradiation, irradiated and non-irradiated MSCs were trypsinized, mixed (0, 25, 50, 75%, and 100% irradiated versus non-irradiated cells), and centrifuged at 300 g for 8 min to obtain pellets of 2 × 105 cells. To induce chondrogenesis, cell pellets were cultured in a chondrogenic medium, containing a DMEM-HG medium (Invitrogen brand Thermo Fisher Scientific), supplemented with 1% ITS (BD, Franklin Lakes, NJ, United States), 1.5 μg/mL fungizone (Invitrogen brand Thermo Fisher Scientific), 50 μg/mL gentamicin (Invitrogen brand, Thermo Fisher Scientific), 1 mM sodium pyruvate (Invitrogen brand, Thermo Fisher Scientific), 40 μg/mL proline (Sigma-Aldrich), 10 ng/mL TGFβ1 (R&D Systems), 0.1 mM ascorbic acid-2-phosphate (Sigma-Aldrich), and 100 nM dexamethasone (Sigma-Aldrich) for 7, 14, or 21 days. The medium was renewed twice a week.
9
Palmitate and High Glucose Stress on HMCs
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The HMCs were revived and cultured using DMEM low glucose medium (Gibco, Shanghai, China) containing 10% fetal bovine serum (Tianhang, Hangzhou, China) in an incubator at 37 °C with 5% CO2. When HMCs grew to 80%, they were inoculated into culture plates and divided into the control, palmitate (PA, 160 μM), HG (HG, 25 mM), and PA (160 μM)+HG (25 mM) group. All groups of cells were treated for 24 h. The treatment environment for HG is the DMEM HG medium (Gibco, Shanghai, China). The PA was prepared as follows. BSA solids were first dissolved in distilled water at 55 °C and configured into a 30% BSA solution. The PA was dissolved in distilled water at 70 °C to make a 27 mM PA stock solution. The above configured BSA and PA solutions were mixed 1:1 to be a 13.5 mM PA working solution.
10
Ferritin-Mediated Cell Uptake Assay
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Human colon adenocarcinoma HT29 cells (ATCC), human embryonic kidney HEK293 cells (ATCC), mouse brain cortex BEND3 cells (ATCC), and mouse embryonic fibroblasts 3HC/10T1/2 cells (ATCC) were maintained in Dulbecco’s modified Eagle’s medium (DMEM HG medium) (Gibco™; Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Bioind) and antibiotics (penicillin 100 UI/mL, streptomycin 100 μg/mL), at 37 °C in 5% CO2. For uptake assays, cells were seeded in a 96-well plate at a density of 2500 cells/100 µL of medium and incubated for 24 h with FTH and FTL containing gold nanoparticles or iron oxides (as loading control). ApoFTH and ApoFTL were used as control. The added ferritin samples never exceed 10% of cell culture volume.
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