Sodium chromate, Triton X-100, demecolcine and potassium chloride (KCl) were purchased from Sigma Chemical (St. Louis, MO). Giemsa stain was purchased from Ricca Chemical Company. (Arlington, TX). Crystal violet, sodium dodecyl sulfate (SDS), and acetic acid were purchased from J.T. Baker (Phillipsburg, NJ). Nitric acid and methanol were purchased from BDH Chemicals (Radnor, PA). Gurr’s buffer, Keratinocyte-SFM, and 4′,6-diamidino-2-phenylindole (DAPI) were purchased from Invitrogen Corporation (Grand Island, NY). Tissue culture dishes, flasks and plasticware were purchased from BD (Franklin Lakes, NJ). Urothelial cell medium, trypsin/EDTA and trypsin neutralizing solution were purchased from ScienCell Research Laboratories (Carlsbad, CA). Penicillin/streptomycin was purchased from Mediatech (Manassas, VA). Paraformaldehyde 4% was purchased from Alfa Aesar (Ward Hill, MA). Phospho-histone H2A.X (Ser139) antibody was purchased from Cell Signaling (Beverly, MA). AlexaFluor 488-conjugated goat anti-rabbit IgG secondary antibody was purchased from ThermoScientific (Rockford, IL).
Trypsin edta
Manufactured by ScienCell
Trypsin/EDTA is a solution used for the detachment and dissociation of adherent cells in cell culture. It contains the proteolytic enzyme trypsin and the chelating agent EDTA, which together function to break down the extracellular matrix and cell-cell adhesions, allowing cells to be easily removed from the culture vessel.
Automatically generated - may contain errors
Lab products found in correlation
Trypsin neutralizing solution, by ScienCell (2 mentions)
Methanol, by Avantor (1 mentions)
Paraformaldehyde 4, by Thermo Fisher Scientific (1 mentions)
Keratinocyte sfm, by Thermo Fisher Scientific (1 mentions)
Phospho histone h2a x ser139 antibody, by Cell Signaling Technology (1 mentions)
Sodium chromate, by Merck Group (1 mentions)
Gurr s buffer, by Thermo Fisher Scientific (1 mentions)
Demecolcine, by Merck Group (1 mentions)
Nitric acid, by Avantor (1 mentions)
Alexa fluor 488 conjugated goat anti rabbit igg secondary antibody, by Thermo Fisher Scientific (1 mentions)
Urothelial cell medium, by ScienCell (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Acetic acid, by Avantor (1 mentions)
Crystal violet, by Avantor (1 mentions)
Sodium dodecyl sulfate (sds), by Avantor (1 mentions)
Tissue culture dishes, by BD (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
Rneasy minelute cleanup kit, by Qiagen (1 mentions)
Rnase free dnase set, by Qiagen (1 mentions)
4 protocols using trypsin edta
1
Immunofluorescent Analysis of DNA Damage
2
RNA Isolation and cDNA Synthesis Protocol
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Total RNA was isolated via the RNeasy kit (Qiagen, Valencia, CA, United States). After aspiration of media from stimulated cultures, adherent cells were washed 3X with warm, sterile PBS. Cells were suspended with trypsin-EDTA (ScienCell) and all enzymatic activity neutralized with the addition of microglial medium containing 5% fetal bovine serum. Buffer RLT (Qiagen) was used to lyse cells. Genomic DNA was then removed by on-column DNA digestion with RNase-Free DNase Set (catalog # 79254, Qiagen). RNA was then concentrated using a the Qiagen RNeasy MinElute Cleanup Kit (catalog # 74204). RNA quality and concentration was assessed by spectrometry (Epoch, Bio-Tek, Winooski, VT, United States). cDNA was synthesized with Qiagen RT2 First Strand Kit (Catalog # 330401) following manufacturer’s instructions.
3
Primary HSCs and HiBECs Culture
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Primary human HSCs and primary human intrahepatic biliary epithelial cells (HiBECs, cholangiocytes) were purchased from ScienCell Research Laboratories (5300 & 4100, respectively). HSCs were cultured in stellate cell medium (ScienCell, 5301) supplemented with 2% fetal bovine serum (FBS), stellate cell growth supplements, and 1% penicillin/streptomycin (P/S). HiBECs were cultured in epithelial cell medium (ScienCell, 4101) supplemented with 2% FBS, epithelial cell growth supplement (ScienCell), and 1% P/S. Cells were grown in flasks precoated with poly-l -lysine and subcultured with trypsin/EDTA and trypsin neutralizing solution (ScienCell, 0103 & 0113, respectively) according to supplier protocol. LX2 cells (Merck, catalog SCC064) were cultured in DMEM, with 10% FCS. In cell culture experiments, supernatants were assayed for CCL24 using ELISA (R&D Systems, catalog DY343).
4
Purification of Human Fetal Astrocytes
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Human fetal astrocytes (ScienCell cat#1800) were cultured on poly-L-lysine coated plates in Astrocyte Media (ScienCell). Media was changed every 2 days and cells were passaged 1:6 when 90% confluent using trypsin-EDTA (ScienCell). 10% fetal bovine serum (FBS, Gibco) was added to media for 6 passages (6 weeks) as previously described to purify astrocyte cultures of neural progenitors (Zhang et al., 2015 (link)). Astrocytes were incubated for 24 h on an orbital shaker plate at 200 rpm as previously described to purify low passage (P2) astrocytes of neural progenitors (Lian et al., 2016 (link)). Mouse embryonic fibroblasts (MEFs) were purchased from MTI GlobalStem and thawed onto poly-L-ornithine (PLO)/laminin (Sigma Aldrich) substrates in high glucose DMEM (Gibco) supplemented with 10% FBS.
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