Islets were isolated from NOR mice. Briefly, the pancreas was injected through the papilla of Vaters with 2.5 mL Collagenase XI solution: Collagenase XI (Sigma-Aldrich, #C7657) dissolved in HBSS buffer supplemented with Ca2+, Mg2+ (CarlRoth, #9119.1) and 0.08% BSA. The harvested pancreas was incubated in a water bath at 37 °C for 15 min. The digestion was stopped by the addition of cold RPMI medium (Corning (Corning, NY, USA), #10–040-CV) supplemented with 10% FBS. Following digestion, the pancreas was transferred through a metallic strainer and washed two times with RPMI. Islet separation was performed with 10 mL of Histopaque-1077 (Sigma-Aldrich, #10771) overlaid with 5 mL RPMI and centrifuged at 850× g for 15 min. Islets are transferred from the gradient with a pipette to a new tube with RPMI. Islets were washed three times with RPMI, resuspended in RPMI with 10% FBS and hand-picked. For treatments, islets were cultured in DMEM low glucose (PAN-Biotech), supplemented with 10% FBS and 1% penicillin/streptomycin, for the indicated time.
Dmem low glucose
Manufactured by PAN Biotech
Sourced in Germany
DMEM low glucose is a cell culture medium that provides essential nutrients for the growth and maintenance of cells in vitro. It contains a lower concentration of glucose compared to standard DMEM, making it suitable for cell lines that are sensitive to higher glucose levels.
Automatically generated - may contain errors
Lab products found in correlation
Mem non essential amino acids solution, by Thermo Fisher Scientific (2 mentions)
Histopaque 1077, by Merck Group (1 mentions)
Rpmi medium, by Corning (1 mentions)
Collagenase xi, by Merck Group (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Biowest (1 mentions)
Dulbecco modified eagle medium (dmem), by Harvard Bioscience (1 mentions)
β mercaptoethanol, by Merck Group (1 mentions)
Penicillin, by Harvard Bioscience (1 mentions)
Streptomycin, by Harvard Bioscience (1 mentions)
Dulbecco modified eagle medium (dmem), by InvivoGen (1 mentions)
L glutamine, by Harvard Bioscience (1 mentions)
Blasticidin, by InvivoGen (1 mentions)
Penicillin, by PAN Biotech (1 mentions)
Puromycin, by InvivoGen (1 mentions)
Streptomycin, by PAN Biotech (1 mentions)
Paclitaxel, by Merck Group (1 mentions)
4 protocols using dmem low glucose
1
Isolation and Culture of Mouse Pancreatic Islets
2
Culturing Human Dermal Fibroblasts and HaCaT Cells
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Human Dermal Fibroblast (HDF) (Lonza, Switzerland) cells were cultured on cultureware coated with 0.1% gelatin (PAN Biotech, Germany) in Dulbecco’s Modified Eagle’s Medium (DMEM) – low glucose (PAN Biotech, Germany) with 10% fetal bovine serum (FBS) (Biowest, France), 1% penicillin–streptomycin (10,000 U/mL) (Gibco, USA), 1% GlutaMAX™ (100 ×) (Gibco, USA), 1% MEM non-essential amino acids solution (100 ×) (Gibco, USA) and 50 mM 2-Phospho-L-ascorbic acid trisodium salt (MilliporeSigma, USA).
HaCaT cells (AddexBio, USA) were cultured on cultureware coated with 0.1% gelatin (PAN Biotech, Germany) in DMEM (Gibco, USA) with 10% FBS (Biowest, France), 1% penicillin–streptomycin (10,000 U/mL) (Gibco, USA), 1% GlutaMAX™ (100 ×) (Gibco, USA) and 1% MEM non-essential amino acids solution (100 ×) (Gibco, USA).
HaCaT cells (AddexBio, USA) were cultured on cultureware coated with 0.1% gelatin (PAN Biotech, Germany) in DMEM (Gibco, USA) with 10% FBS (Biowest, France), 1% penicillin–streptomycin (10,000 U/mL) (Gibco, USA), 1% GlutaMAX™ (100 ×) (Gibco, USA) and 1% MEM non-essential amino acids solution (100 ×) (Gibco, USA).
3
Cell Culture Conditions for Various Cell Lines
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HEK293T, human HT1080, murine NIH3T3, murine SC-1 cells, and their derivatives were cultured in DMEM (Biochrom, Berlin, Germany) with 10% heat-inactivated fetal bovine serum (FBS) (PAN Biotech, Aidenbach, Germany), 100 U/mL penicillin, 100 μg/mL streptomycin, and 1 mM sodium pyruvate (all from PAA, Coelbe, Germany). In the case of human and murine RFP657.Tet2 and RFP657.Tet2+sgRNA.Tet2 reporter cells, DMEM was supplemented with 1.5 μg/mL puromycin (InvivoGen, Toulouse, France). Murine and human RFP657.Tet2+SpCas9 reporter cells were cultured in the presence of 1.5 μg/mL puromycin and 20 μg/mL blasticidin (InvivoGen). Human Jurkat cells were cultured in RPMI medium 1640 (PAN Biotech) supplemented with 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, and 1 mM sodium pyruvate. Primary NUFF cells were purchased (passage 9 after isolation from healthy donors; Amsbio, Abingdon, United Kingdom) and cultivated in DMEM low glucose (PAN Biotech) supplemented with 15% heat-inactivated FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, 2 mM L-glutamine (Biochrom), 1% minimal essential medium (MEM) nonessential amino acids solution (Gibco/Thermo Fisher Scientific, Schwerte, Germany) and 100 μM β-mercaptoethanol (Sigma-Aldrich, Munich, Germany).
4
MTT Assay for Cyanobacterial Cytotoxicity
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(3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide -Sigma-Aldrich, USA) kit was used for evaluation of the cytotoxic activity of cyanobacterial extracts on Human cervix epithelioid carcinoma (HeLa ATCC CCL-2). Cells were seeded in a 96-well microplate, 5000 cells/well in a 200 µL culture medium (DMEM low glucose -PAN biotech, Germany) and kept at 37 °C and 5% CO 2 /95% air for 24 hours. After the incubation period, each well was supplemented with 50 µL cyanobacterial extracts at a final concentration of 500.0, 250.0, 125.0, 62.5, 31.2 or 15.6 µg/mL. For the fractions eluted from LH-20 column chromatography, the final concentration in each well was 20.0 µg/mL. Each concentration was tested in three parallels, and the experiment was repeated three times. Paclitaxel (Sigma-Aldrich, USA) was used as a positive control at concentrations ranging from 0.003 µg/mL to 30 µg/mL. After 48 hours, cells were dyed with MTT, and the optical density was measured using a Microplate reader SpectraMax Plus384 (GMI, USA) at two wavelengths of 570 nm and 690 nm [15] . The IC 50 values (the concentration of extract at which 50% of cell growth was inhibited) were calculated by Excel and GraphPad Prism 8 software.
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