PCAs were prepared from neonatal C57BL/6J mice as described elsewhere [35 (link), 41 (link)]. Briefly, the isolated forebrain was minced and incubated with DNase I (FUJIFILM Wako Pure Chemical) and trypsin (Thermo Fisher Scientific) for dissociation. The dissociated tissues were suspended in Dulbecco’s modified Eagle medium/Nutrient Mixture F-12 (DMEM/F12; Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich, St. Louis, MO, USA) and 1% penicillin/streptomycin (PS; Thermo Fisher). The suspended cells were seeded in poly-l -lysine (PLL; ScienCell, Carlsbad, CA, USA)-coated 75-mm2 flasks (10–15 × 106 cells/flask). The plated cells were cultured in a 5% CO2 incubator at 37°C. Every 8–12 days, the confluent cells were shaken for 10 min to separate them from microglial cells, released from the flasks with trypsin, and seeded onto non-coated flasks. These cells were seeded onto non-coated 8-well chamber slides (2.5 × 104 cells/well) and evaluated with immunocytochemistry for anti-glial fibrillary acidic protein (GFAP) antibody (Abcam).
Poly l lysine pll
Manufactured by ScienCell
Sourced in United States
Poly-L-Lysine (PLL) is a synthetic, positively charged amino acid polymer. It is commonly used to promote cell adhesion and growth on cell culture surfaces.
Automatically generated - may contain errors
Lab products found in correlation
Dmem f12, by Thermo Fisher Scientific (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Dnase 1, by Fujifilm (1 mentions)
Penicillin streptomycin p s, by Thermo Fisher Scientific (1 mentions)
Matrigel, by Corning (1 mentions)
Gelatin, by Merck Group (1 mentions)
Huvecs, by ScienCell (1 mentions)
Penicillin streptomycin solution, by ScienCell (1 mentions)
Accutase, by Innovative Cell Technologies (1 mentions)
Egm 2mv, by Lonza (1 mentions)
Stem cellbanker, by Nippon Zenyaku Kogyo (1 mentions)
Phenazine methosulfate pms, by Merck Group (1 mentions)
3 4 5 dimethylthiazol 2 yl 5 3 carboxymethoxyphenyl 2 4 sulfophenyl 2h tetrazolium mts, by Merck Group (1 mentions)
Tnf α elisa kit, by BD (1 mentions)
Fetal bovine serum (fbs), by American Type Culture Collection (1 mentions)
Carvacrol, by Merck Group (1 mentions)
Peptidoglycan pgn, by Cedarlane (1 mentions)
5 protocols using poly l lysine pll
1
Astrocyte Culture from Neonatal Mouse Forebrain
2
Engineered 3D Cell Culture Microenvironments
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35 mm, low walled petri dishes (μ-Dish 35 mm low Grid-500, Ibidi) were prepared by coating the surface with one of the following materials: 0.1% w/v gelatin (from porcine skin, gel strength 300 Type A, Sigma) in sterile water and incubated at room temperature for 5 minutes, Matrigel (Corning) diluted 1:80 in DMEM/F12 and incubated at 37 C for 1 hour, or 2 μg/mL Poly-L-Lysine (PLL, ScienCell) in sterile water and incubated at 37 C for 1 hour. Excess solution was aspirated and replaced with 1.5 mL of the cells’ complete medium minus FBS. PLL coated dishes requires two washes with sterile water before the addition of cell culture medium. To create three-dimensional stacks of collagen, the complete medium was aspirated from the dish following the completion of printing. An additional 25 uL of collagen I was gently pipetted onto the surface and incubated at 37 C and 5% CO2 for 15 minutes. Fresh medium was added to the dish and the printing resumed on the new layer of collagen.
3
Culturing Human Umbilical Vein Endothelial Cells
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Human umbilical vein endothelial cells (HUVECs, #8000), endothelial cell medium (ECM, #1001), fetal bovine serum (FBS, #0025), penicillin/streptomycin solution (P/S, #0503), endothelial cell growth supplement (ECGS,#1052) and Poly-L-Lysine (PLL, #0403) were all purchased from ScienCell Research Laboratories (San Diego, CA). HUVECs were grown on PLL coated culture plate in ECM supplemented with 5% FBS, 1% P/S and 1% ECGS.
4
Cryopreservation of Retinal Cell Types
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Original vials of primary Human Retinal Microvascular Endothelial Cells (HRMVECs, Cell Systems, ACBRI 181) were obtained at passage 3 (P3), Human Retinal Pericytes (HRPs, Cell Systems, ACBRI 183) at P3 and Human Retinal Astrocytes (HRAs, ScienCell, 1870) at P1. The list of all cells used in this study is provided in Supplementary Table I . HRMVECs were cultured in T175 flasks coated for 1 h at 37 °C with 1× Attachment Factor Protein (Gibco, S1006100), in EGM-2 MV Microvascular Endothelial Cell Growth Medium-2 BulletKit (EGM-2 MV, Lonza, CC-3202). HRPs and HRAs were cultured in T175 flasks coated for 1 h at room temperature (RT) with 15 μg ml−1 Poly-L-Lysine (PLL, ScienCell, 0403), in Pericyte Medium (PM, ScienCell, 1201) or Astrocyte Medium (AM, ScienCell, 1801), respectively. Cells were harvested with Accutase (Innovative Cell Technologies, AT104). We prepared working stocks by expanding HRMVECs and HRPs to P5 and HRAs to P3, resuspending 1 × 106 cells ml-1 in STEM-CELLBANKER (Nippon Zenyaku Kogyo, 11897) freezing medium and freezing 5 × 105 cells per vial. New frozen vials were used for each experiment.
5
Evaluation of Anti-inflammatory Effects of Carvacrol
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Carvacrol (≥98%, food-grade), Dulbecco’s phosphate-buffered saline (DPBS), dimethyl sulfoxide (DMSO), lipoteichoic acid (LTA), 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS), and phenazine methosulfate (PMS) were obtained from Sigma-Aldrich (Oakville, ON, Canada). Peptidoglycan (PGN) was obtained from Cedarlane Laboratories (Burlington, ON, Canada). Human tonsil epithelial cells (HTonEpiCs), tonsil epithelial cell medium, tonsil epithelium cell growth supplement, poly-L-lysine (PLL), trypsin neutralization solution (TNS), and trypsin-ethylenediamine tetra acetic acid (0.25%) solution (TE) were purchased from ScienCell Research Laboratory (San Diego, CA, USA). Fetal bovine serum (FBS) was purchased from American Type Culture Collection (ATCC) (Manassas, VA, USA). 7-Aminoactinomycin D (7-AAD) stain was purchased from BioLegend, Inc. (San Diego, CA, USA). Enzyme-linked immunosorbent assay (ELISA) kits were purchased from different manufacturers: IL-6 and TNF-α ELISA kits were purchased from BD Biosciences (Mississauga, ON, Canada); human ENA-78, GCP-2, and human COX-2 ELISA kits were purchased from RayBiotech, Inc. (Norcross, GA, USA); a human BD-2 ELISA kit was purchased from PromoCell GmbH (Sickingenstraße, Heidelberg, Germany); and PGE2 and IL-8 kits were purchased from Invitrogen (Nepean, ON, Canada).
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