The viability staining was performed with the stimulated chondrocytes, cultivated on Poly-L-lysin (Biochrom AG, Darmstadt, Germany) coated cover slides. The cells were incubated for 3 min at room temperature in a mixture of fluorescein diacetate (Sigma-Aldrich, Munich, Germany), which stains viable cells green, and propidiumiodide (Carl Roth GmbH, Karlsruhe, Germany), coloring dead cells red. The green and red fluorescence were visualized using confocal laser scanning microscopy (Leica TCS SPEII and DMi8, Wetzlar, Germany). The images of chondrocytes were analyzed using the Image Processing software ImageJ (US National Institutes of Health, Bethesda, MD, USA). The area covered by living cells was calculated in relation to the total area colonized by cells.
Poly l lysin
Manufactured by Harvard Bioscience
Sourced in Germany
Poly-L-lysine is a synthetic polymer composed of the amino acid L-lysine. It is commonly used as a coating for cell culture surfaces to enhance cell attachment and growth. Poly-L-lysine increases the positive charge on the surface, which helps cells adhere more effectively.
Automatically generated - may contain errors
Lab products found in correlation
Fluorescein diacetate, by Merck Group (2 mentions)
Nifuroxazide, by Merck Group (2 mentions)
Sb 216763, by Adipogen (2 mentions)
Lithium chloride (licl), by Merck Group (2 mentions)
Lipopolysaccharides (lps), by Merck Group (2 mentions)
Propidium iodide, by Carl Roth (1 mentions)
Tcs spe 2, by Leica (1 mentions)
Axioskop 40, by Zeiss (1 mentions)
Colorview 2, by Olympus (1 mentions)
Ethidium bromide, by Carl Roth (1 mentions)
Phosphate buffered saline (pbs), by Harvard Bioscience (1 mentions)
Paraformaldehyde pfa, by Santa Cruz Biotechnology (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Harvard Bioscience (1 mentions)
Non essential amino acids (neaa), by Harvard Bioscience (1 mentions)
Trypsin, by Harvard Bioscience (1 mentions)
Ascorbic acid, by Merck Group (1 mentions)
Penicillin, by Harvard Bioscience (1 mentions)
Streptomycin, by Harvard Bioscience (1 mentions)
Amphotericin b, by Harvard Bioscience (1 mentions)
T25 cell culture flasks, by Sarstedt (1 mentions)
7 protocols using poly l lysin
1
Chondrocyte Viability Quantification
2
Tenocyte Viability Quantification
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Tenocytes seeded on Poly-L-lysin (Biochrom)-coated cover slides and stimulated with the agents for 72 h were incubated in a mixture of 5 µL/mL fluorescein diacetate (Sigma-Aldrich, 3 mg/mL dissolved in acetone (stock solution)) and 1 µg/mL ethidium bromide (Carl-Roth, Karlsruhe, Germany) diluted in 1 mL phosphate-buffered saline (PBS) for 10 min. The green (vital) or red (dead) cell fluorescence was visualized using fluorescence microscopy (Axioskop 40, Carl Zeiss, Jena, Germany) using a digital camera (Color View II, Olympus, Shinjuku, Japan). Three microscopic fields of each treatment group in each independent experiment were evaluated using ImageJ for vital and dead cells.
3
Preparing Coated Glass Coverslips for Cell Culture
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For analyzing the expression profile of marker proteins, glass cover slips were coated. They were washed twice with PBS (Biochrom, Berlin, Germany). After washing with sterile A. dest., the glass cover slips were placed individually in sterile petri dishes and covered with poly-L-lysin solution (1:100 in PBS; both Biochrom) for 20 min at RT. Subsequently, the coated glass cover slips were rinsed with sterile A. dest. and dried at RT for 4 h. Until further use they were stored at 4 °C.
For vitality assay and immunofluorescence analysis ACL ligamentocytes were seeded on the coated glass cover slips with an initial density of 1 × 104 cells/cm² and allowed to adhere for another 24, 48, and 72 h. Before respective staining procedures, medium was removed and cells washed in PBS. For vitality analysis the glass cover slips were immediately transferred to Live/Dead assay, while for performing the immunofluorescence analysis or histological staining, cells were fixed in 4% paraformaldehyde (PFA, Santa Cruz Biotechnology Inc., Texas, USA) for 15 min and stored at 4 °C immersed in PBS.
For vitality assay and immunofluorescence analysis ACL ligamentocytes were seeded on the coated glass cover slips with an initial density of 1 × 104 cells/cm² and allowed to adhere for another 24, 48, and 72 h. Before respective staining procedures, medium was removed and cells washed in PBS. For vitality analysis the glass cover slips were immediately transferred to Live/Dead assay, while for performing the immunofluorescence analysis or histological staining, cells were fixed in 4% paraformaldehyde (PFA, Santa Cruz Biotechnology Inc., Texas, USA) for 15 min and stored at 4 °C immersed in PBS.
4
Isolation and Expansion of ACL Ligamentocytes
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ACL samples (one female donor, age 40), were harvested in accordance with the institutional ethical committee of the Charité-Universitätsmedizin Berlin, Campus Benjamin Franklin (ethical approval number EA4-033-08, approval date: 22 May 2008). Surrounding connective tissue, including the synovial membranes, was removed. The pure ligament tissue was cut into 1–2 mm2 slices and incubated in T25 cell culture flasks (Sarstedt, Nümbrecht, Germany) with growth medium (Ham’s F-12/Dulbecco’s Modified Eagle’s [DMEM] Medium 1:1) containing 10% fetal calf serum (FCS), 10,000 IU/mL penicillin/10,000 µg/mL streptomycin, 2.5 µg/mL amphotericin B, non-essential amino acids (all from Biochrom, Berlin, Germany) and 25 µg/mL ascorbic acid (Sigma-Aldrich, Munich, Germany) at 37 °C and 5% CO2. After 1–2 weeks, the ACL ligamentocytes started to migrate from the tissue slices. Subsequently, ligamentocytes were harvested using 0.05% trypsin/0.02% EDTA (Biochrom) and expanded in T75 and T175 culture flasks. For analyzing the expression of marker protein profile on protein level, ACL ligamentocytes were seeded on poly-L-lysin (Biochrom) coated cover slips at 1 × 104 cells/cm².
5
Protein Expression Profiling of Fibroblasts
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For analyzing the marker expression profile on protein level, cover slips were washed twice with phosphate buffered saline (PBS, Biochrom AG). After rinsing with sterile A. dest., the cover slips were placed in sterile petri dishes and covered with a poly-l -lysin solution (1:100 in 1x PBS; both Biochrom AG) for 20 min at room temperature (RT). Subsequently, the coated cover slips were rinsed with sterile A. dest. and dried at RT for 4 h. Until further use, they were stored at 4 °C.
For the vitality assay and immunofluorescence analysis, ITB (passages 1–3) as well as ACL fibroblasts (passages 1–5) were seeded on the coated cover slips with an initial density of 1 × 104 cells/cm2 and allowed to adhere for another 24 h. Before the respective staining procedures, the medium was removed and the cells were washed in 1× PBS. For the vitality analysis, the cover slips were immediately transferred to live/dead assay, while for performing the immunofluorescence analysis, the cells were fixed in 4% PFA for 15 min and stored at 4 °C.
For the vitality assay and immunofluorescence analysis, ITB (passages 1–3) as well as ACL fibroblasts (passages 1–5) were seeded on the coated cover slips with an initial density of 1 × 104 cells/cm2 and allowed to adhere for another 24 h. Before the respective staining procedures, the medium was removed and the cells were washed in 1× PBS. For the vitality analysis, the cover slips were immediately transferred to live/dead assay, while for performing the immunofluorescence analysis, the cells were fixed in 4% PFA for 15 min and stored at 4 °C.
6
Modulating Cell Activation Pathways
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Plasticware was coated with poly-L-lysin (diluted 1:20 in distilled water; Biochrom, Merck) or collagen A (0.5 mg/ml Biochrom, Merck) overnight at 4 C followed by washes with 1xDPBS (Gibco™, Thermo Fisher Scientific). Cells were activated with LPS (1 μg/ml; Sigma-Aldrich) or human Peprotech) . Lithium chloride (10 mM; Sigma-Aldrich), SB-216763 (10 μM; AdipoGen), and nifuroxazide (10 μM; Sigma-Aldrich) were applied 30 min before activation.
7
Isolation and Culture of Human Microglia
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Plasticware was coated with with poly-L-lysin (diluted 1:20 in distilled water; Biochrom, Merck) or collagen A (0.5 mg/ml Biochrom, Merck) overnight at 4°C followed by washes with 1xDPBS (Gibco TM , Thermo Fisher Scientific). Cells were activated with LPS (1 µg/ml; Sigma-Aldrich) or human IFN-γ (200 ng/ml; Peprotech). Lithium chloride (10 mM; Sigma-Aldrich), SB-216763 (10 µM; AdipoGen), and nifuroxazide (10 µM, Sigma-Aldrich) were applied 30 min before activation.
For the isolation of human microglia from surgical brain specimens, we essentially followed a previously published protocol (Rustenhoven et al., 2016) . Briefly, blood vessels and meninges were removed from resected brain tissue using a dissection microscope (Zeiss Stemi DV4). Tissue was weighed, diced and dissociated using the Neural Tissue Dissociation Kit (P) Miltenyi Biotec) . Microglia were cultured as adherent cells in poly-L-lysin coated T75 tissue culture flasks in microglia culture medium containing DMEM/F12 (Gibco TM , Thermo Fisher Scientific), 10% fetal calf serum (Biochrom, Merck) and 1X penicillin-streptomycin solution (Biochrom, Merck).
Purity of cultures was verified by immunostaining (Figure S2).
For the isolation of human microglia from surgical brain specimens, we essentially followed a previously published protocol (Rustenhoven et al., 2016) . Briefly, blood vessels and meninges were removed from resected brain tissue using a dissection microscope (Zeiss Stemi DV4). Tissue was weighed, diced and dissociated using the Neural Tissue Dissociation Kit (P) Miltenyi Biotec) . Microglia were cultured as adherent cells in poly-L-lysin coated T75 tissue culture flasks in microglia culture medium containing DMEM/F12 (Gibco TM , Thermo Fisher Scientific), 10% fetal calf serum (Biochrom, Merck) and 1X penicillin-streptomycin solution (Biochrom, Merck).
Purity of cultures was verified by immunostaining (Figure S2).
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