Mouse primary astrocytes were isolated from naïve P0 to P1 neonatal C57/BL6 mice cortices as previously described by Chen et al. [84 (link)], with minor modifications [85 (link),86 (link)]. Briefly, a mixed glial culture isolated from neonatal mice was grown for 8 days in Dulbecco’s modified Eagle’s medium (DMEM) low glucose (Biological Industries, Kibbutz Beit-Haemek, Israel) supplemented with 5% fetal bovine serum (Biological Industries, Kibbutz Beit-Haemek, Israel), 1 mM sodium pyruvate (Biological Industries, Kibbutz Beit-Haemek, Israel), 1 mM L-glutamine (Sigma–Aldrich, Rehovot, Israel) and 0.6% Gentamycin Sulfate (Biological Industries, Kibbutz Beit-Haemek, Israel). Culture medium was replaced every 2 days. After 8 days, microglia were detached by 30 min shaking at 140 rpm using an orbital shaker. After medium was removal, a new fresh culture medium was added, and OPCs at the top of the astrocyte monolayer were detached by shaking for 18 h at 200 rpm. Media was replaced and astrocytes were grown for further 7 days. Cells were detached from flasks using TrypLE (Thermo Fisher Scientific, Waltham, MA, USA). All cultures expressed high level of GFAP (mean of 96.6% GFAP+ cells, Figure S1 ).
Low glucose dmem
Manufactured by Sartorius
Sourced in Israel
Low glucose DMEM is a cell culture medium formulated with a reduced concentration of glucose. It is designed to support the growth and maintenance of various cell lines, particularly those with sensitivity to high glucose levels. The reduced glucose content aims to mimic physiological conditions and may be beneficial for specific experimental requirements.
Automatically generated - may contain errors
Lab products found in correlation
Sodium pyruvate, by Sartorius (4 mentions)
Gentamycin sulfate, by Sartorius (4 mentions)
L glutamine, by Merck Group (3 mentions)
Fetal bovine serum (fbs), by Sartorius (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Stempro 34, by Thermo Fisher Scientific (3 mentions)
Penicillin, by Sartorius (3 mentions)
Streptomycin, by Sartorius (3 mentions)
L glutamine, by Sartorius (3 mentions)
Nystatin, by Sartorius (3 mentions)
Stempro 34 nutrient, by Sartorius (3 mentions)
High glucose dmem, by Sartorius (2 mentions)
Fetal bovine serum (fbs), by Cytiva (2 mentions)
Tryple, by Thermo Fisher Scientific (1 mentions)
Glutamine, by Merck Group (1 mentions)
Pdgf aa, by R&D Systems (1 mentions)
Fibroblast growth factor 2 (fgf2), by R&D Systems (1 mentions)
Dulbecco modified eagle medium (dmem), by Sartorius (1 mentions)
Trypsin edta, by Sartorius (1 mentions)
Mcf 7, by Sartorius (1 mentions)
13 protocols using low glucose dmem
1
Isolation of Mouse Primary Astrocytes
2
Effects of Postoperative Care Agents on Gingival Fibroblasts
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HGFs were obtained from Erciyes University Betul-Ziya Eren
Genome and Stem Cell Center (Kayseri, Turkey) and cultured
in DMEM-Low Glucose (Biological Industries, Kibbutz Beit
Haemek, Israel) supplemented with 10% fetal bovine serum
(FBS), 100 IU/mL penicillin and streptomycin, and incubated
at 37°C in a 5% humidified CO2 atmosphere. An ethical
consideration was not required.
The samples of the study were divided into the four
groups to compare effects of postoperative care agents. The
groups were; chlorhexidine applied group (CHX), octenidine
dihydrochloride applied (OCT) group, hyaluronic acid (HA)
applied and nothing (contol group) were applied on human gingival fibroblasts’ cell.
Genome and Stem Cell Center (Kayseri, Turkey) and cultured
in DMEM-Low Glucose (Biological Industries, Kibbutz Beit
Haemek, Israel) supplemented with 10% fetal bovine serum
(FBS), 100 IU/mL penicillin and streptomycin, and incubated
at 37°C in a 5% humidified CO2 atmosphere. An ethical
consideration was not required.
The samples of the study were divided into the four
groups to compare effects of postoperative care agents. The
groups were; chlorhexidine applied group (CHX), octenidine
dihydrochloride applied (OCT) group, hyaluronic acid (HA)
applied and nothing (contol group) were applied on human gingival fibroblasts’ cell.
3
Isolation and Culture of Oligodendrocyte Precursor Cells
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Primary OPC cultures were isolated from naïve P0 to P1 neonatal C57/BL6 mice cortices as previously described by Chen et al. (2007 (link)), with minor modifications (Barateiro & Fernandes, 2014 (link)). Briefly, a mixed glial culture isolated from neonatal mice was grown for 8 days in Dulbecco's modified Eagle's medium (DMEM) low glucose (Biological Industries, Israel) supplemented with 5% fetal bovine serum (Biological Industries, Israel), 1 mM sodium pyruvate (Biological Industries, Israel), 1 mM L‐glutamine (Sigma‐Aldrich, Israel) and 0.6% Gentamycin Sulfate (Biological Industries, Israel). Culture medium was replaced every 2 days. After 8 days, microglia were detached by 30 min shaking at 140 rpm using an orbital shaker. After medium was removal, a new fresh culture medium was added, and OPCs at the top of the astrocyte monolayer were detached by shaking for 18 h at 200 rpm. Cells were plated in DMEM/F‐12 (Rhenium, Israel) supplemented with 1% B27 supplement (Rhenium, Israel) and 0.6% Gentamycin Sulfate (Biological Industries, Israel). Fibroblast growth factor‐2 (FGF‐2; 20 ng/ml; R&D systems) and Platelet‐derived growth factor (PDGF‐AA; 20 ng/ml; R&D systems) were added to cultures daily unless otherwise specified. Timeline is depicted in Figure S1 a.
4
Astrocytes Exposed to NMO Sera
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Cells were seeded in plates for 24 h. Then, media was replaced for DMEM low glucose (Biological Industries, Kibbutz Beit-Haemekm Israel) supplemented with 1 mM sodium pyruvate (Biological Industries, Kibbutz Beit-Haemek, Israel), 1 mM L-glutamine (Sigma–Aldrich, Rehovot, Israel) and 0.6% Gentamycin Sulfate (Biological Industries, Kibbutz Beit-Haemek, Israel). Human sera of either NMO patients or HCs (20% of media) were added into mouse primary astrocytes media for 48 h. Then, DNA damage response was evaluated using anti-H2AX (Cat# sc-517348, Santa Cruz biotechnology Inc., Dallas, TX, USA, 1:100). Each assay was repeated independently at least three times.
5
Cell Culture and Seeding on Micropatterns
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Cell lines were obtained from commercial suppliers (MCF7, ATCC® HTB-22™ and MDA MD 231, ATCC® CRM-HTB-26™). MCF7 cells were cultured in DMEM low glucose (Biological Industries, Beit-Haemek, Israel) supplemented with 10% FBS, 100 U/mL1 penicillin, and 100 μg/mL1 streptomycin. MDAMB231 cells were cultured in DMEM (Biological Industries, Beit-Haemek, Israel) supplemented with 10% FBS, 100 U/mL1 penicillin, and 100 μg/mL1 streptomycin. Cells were grown in cell culture flasks until 90% confluency and detached using 0.05% Trypsin EDTA (Biological Industries, Israel). The cell suspension is counted and seeded on the micropatterns with a density of 5 × 104 cells/mL1 (5 × 104 cells/well), and the chips were cultured in a Petri dish at 37 °C, 5% CO2. For controls, the same number of cells was seeded into 24-well plates.
6
Apoptosis of Astrocytes by NMO Sera
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Cells were seeded in plates for 24 h. Then, media was replaced for DMEM low glucose (Biological Industries, Kibbutz Beit-Haemek, Israel) supplemented with 1 mM sodium pyruvate (Biological Industries, Kibbutz Beit-Haemek, Israel), 1 mM L-glutamine (Sigma–Aldrich, Rehovot, Israel) and 0.6% Gentamycin Sulfate (Biological Industries, Kibbutz Beit-Haemek, Israel). Human sera of either NMO patients or HCs (20% of media) were added into mouse primary astrocytes media for 72 h. Apoptosis was assessed by Annexin V detection kit (Cat# BG-62700, EMELCA Bioscience, Clinge, The Netherlands). All fluorescence-activated cell sorting (FACS) samples were analyzed in a Beckman coulter FC500 apparatus using the CXP software. Each assay was repeated independently at least three times.
7
3T3-L1 Preadipocyte Assay Protocol
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3T3-L1 mouse preadipocytes were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). High glucose DMEM, low glucose DMEM, Pen-Strep solution (P/S), insulin, certified fetal bovine serum (FBS), special newborn calf serum (NBCS), and phosphate buffered saline (PBS) were purchased from Biological Industries (Shanghai, China). The glucose test kit was purchased from Rongsheng Biotech Co., Ltd. (Shanghai, China). α-Glucosidase (solid), 3,5-dinitrosalicylic acid, p-nitrophenyl α-D-glucopyranoside (PNPG), and ascorbic acid were purchased from Yuanye Biotech Co., Ltd. (Shanghai, China). Acarbose and rutin were obtained from Solarbio (Beijing, China). CellTiter 96® AQueous One Solution Reagent (Promega Corporation, Madison, WI, USA). DPPH, ABTS, and FRAP detection reagents were purchased from Suzhou Comin Biotechnology Co., Ltd. (Jiangsu, China). Sodium nitrite, aluminum nitrate, sodium carbonate, and sodium hydroxide were purchased from MACKLIN (Shanghai, China).
8
Transient Transfection of U2OS Cells
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Human U2OS E3 and E6 cells [24 (link)] were maintained in low glucose DMEM (Biological Industries, Beit-Haemek, Israel), supplemented with 10% fetal bovine serum (HyClone Laboratories, Logan, UT). For transient transfections, cells were transfected with 1–5 μg of plasmid DNA and 40 μg of sheared salmon sperm DNA (Sigma) when using electroporation (Gene Pulser Xcell, Bio-Rad).
For overexpression experiments, the PolyJet reagent (SignaGen) was used. Cells were induced to express the E3 and E6 genes by addition of 1 μg/ml doxycycline (Sigma). Transgenic U2OS E3 and E6 cell lines with stable integration of BACs carrying C-terminally tagged SC35 (SRSF2), SRp75 (SRSF4), SRp40 (SRSF5), SRp55 (SRSF6), 9G8 (SRSF7), U1-70K, U2AF65, and PRP8, were generated as described [26 (link)]. For splicing inhibition, cells were treated for 6 hrs with Pladienolide B (10 μM, Santa Cruz).
For overexpression experiments, the PolyJet reagent (SignaGen) was used. Cells were induced to express the E3 and E6 genes by addition of 1 μg/ml doxycycline (Sigma). Transgenic U2OS E3 and E6 cell lines with stable integration of BACs carrying C-terminally tagged SC35 (SRSF2), SRp75 (SRSF4), SRp40 (SRSF5), SRp55 (SRSF6), 9G8 (SRSF7), U1-70K, U2AF65, and PRP8, were generated as described [26 (link)]. For splicing inhibition, cells were treated for 6 hrs with Pladienolide B (10 μM, Santa Cruz).
9
Culturing RBL-MRGPRX2 and LAD-2 Mast Cells
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RBL-MRGPRX2 cells were generated as previously described [24 (link)]. RBL-MRGPRX2 cells and their parental RBL-2H3 (herein referred to as RBL) counterparts were maintained at 37 °C, in a humidified incubator with 5% CO2, in adherent cell cultures in low-glucose DMEM (cat # 01-050-1A, Biological Industries, Beit-Haemek, Israel) supplemented with 10% FBS (cat # 12657, GIBCO, Grand Island, NY, USA), 2 mM L-Glutamine (cat # 03-020-1A, Biological Industries), 100 μg/mL streptomycin and 100 U/mL penicillin and 12.5 U/mL nystatin (Biological Industries, Beit-Haemek, Israel), except for RBL-MRGPRX2 cells that were additionally supplemented with 1 mg/mL of G418 (cat # A1720, Sigma Aldrich, St Louis, MO, USA). LAD-2 cells (a kind gift from Dr. A.S. Kirshenbaum and Dr. D. Metcalfe, Laboratory of Allergic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA) were cultured in StemPro-34 (cat # 10640-019, GIBCO) supplemented with 1× StemPro-34 Nutrient, 2 mM L-Glutamine (cat # 03-020-1A, Biological Industries), 100 U/mL penicillin and 100 μg/mL streptomycin (cat # 03-032-1B, Biological Industries), and 100 ng/mL hSCF (cat # 300-07, Peprotech, Rocky Hill, NJ, USA).
10
MRGPRX2 Expressing RBL and LAD-2 Cell Lines
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RBL cells stably expressing N-terminally tagged hemagglutinin (HA) human MRGPRX2 (RBL-MRGPRX2) were previously described [12 (link)]. Cells were maintained as adherent cell cultures in low glucose DMEM (cat no. 01-050-1A, Biological Industries, Beit-Haemek, Israel) supplemented with 10% FBS (cat no. 12657, GIBCO, Grand Island, NY, USA), 2 mM L-Glutamine (cat no. 03-020-1A, Biological Industries), 100 μg/mL streptomycin, and 100 U/mL penicillin, 12.5 U/mL nystatin (cat no. 03-032-1B, Biological Industries), and 1 mg/mL of G418 (cat no. A1720, Sigma Aldrich, St. Louis, MO, USA), at 37 °C in a humidified incubator with 5% CO2. LAD-2 cells (a kind gift from Dr. A.S. Kirshenbaum and Dr. D. Metcalfe, Laboratory of Allergic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA) were cultured in StemPro-34 (cat no. 10640-019, GIBCO) supplemented with 1× StemPro-34 Nutrient, 2 mM L-Glutamine (cat no. 03-020-1A, Biological Industries), 100 U/mL penicillin and 100 μg/mL streptomycin (cat no. 03-032-1B, Biological Industries), and 100 ng/mL hSCF (cat no. 300-07, Peprotech, Rocky Hill, NJ, USA).
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