As described previously with modification [7 (link)], mucosal tissue was digested overnight with 0.04% trypsin solution (Life Technologies, Carlsbad, CA, USA) with 19.25 μg/mL of gentamicin (Life Technologies) and 0.765 μg/mL of fungizone (Life Technologies) at room temperature, and transferred into 0.0125% trypsin-inhibitor (Life Technologies). Dissociated oral keratinocytes were resuspended in a chemically defined culture system, complete Epilife (Life Technologies) and seeded into one T-25 flask. For serial cultures, cells were detached in 0.025% trypsin/EDTA (Life Technologies). For analysis, monolayer culture cells were fed adding 30mL of Epilife/150mm Petri culture dish every other day and collected after detachment in Enzyme Free Cell Dissociation Solution (Millipore, Billerica, MA, USA). ePUKs culture was previously described [2 (link)]. The cells were fed adding 60mL of medium/150 mm Petri culture dish, every 24 hour. At confluence, the monolayers continued to proliferate; pushing keratinocytes into the overlying medium and the cells in suspension were collected as ePUKs. The monolayer cells underlying ePUKs culture were collected after detachment in Enzyme Free Cell Dissociation Solution (Millipore).
Enzyme free cell dissociation solution
Manufactured by Merck Group
Sourced in United States
Enzyme-free cell dissociation solution is a reagent used to gently detach adherent cells from cell culture surfaces without the use of enzymes. It is a balanced salt solution designed to facilitate the dissociation of cells while maintaining their viability and function.
Automatically generated - may contain errors
Lab products found in correlation
Facscalibur, by BD (3 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (3 mentions)
Phosstop, by Roche (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Firefly d luciferin, by Nanolight Technology (2 mentions)
Endothelin 1 et 1, by Bio-Techne (2 mentions)
Dulbecco s phosphate buffered saline, by Corning (2 mentions)
Trypsin, by Corning (2 mentions)
Fluorobrite dmem, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Thrombin, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Corning (2 mentions)
Ym 254890, by Adipogen (2 mentions)
Phosstop phosphatase inhibitor, by Merck Group (1 mentions)
Edta free protease inhibitor, by Merck Group (1 mentions)
Colorimetric dc protein assay, by Bio-Rad (1 mentions)
Analysis software, by FlowJo (1 mentions)
Facsverse, by BD (1 mentions)
25 protocols using enzyme free cell dissociation solution
1
Isolation and Culture of Oral Keratinocytes
2
Thioglycollate-Induced Peritoneal Inflammation
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Mice were intraperitoneally administrated with 500 µl of a 4% TG solution (DIFCO Laboratories). After 72 h or the indicated time periods, ice-cold PBS (4 ml) was injected into the peritoneal cavity and 2.5 ml of lavage was collected. The peritoneal cells were washed once with ice-cold PBS and subjected to a flow cytometric analysis. For the analysis of the peritoneal cell recruitment, either BSA or the AGEs (10 mg/kg/day) was daily provided by intravenous administration starting 24 h before the TG injection and continuing until the mice were sacrificed. For the experiments under the inhibitory condition of Plg activation, TXA (Sigma) was administrated in the drinking water at 20 mg/ml 48 h prior to thioglycollate injection and throughout the experiment. For isolation of the peritoneal macrophages, peritoneal exudate cells collected 72 h after the TG injection were incubated in RPMI-1640 for 2 h at 37 °C, wash twice with PBS, and the adherent cells (peritoneal macrophages) were collected using an enzyme-free cell dissociation solution (Sigma).
3
Cell Lysis and Protein Extraction
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Cells were seeded at 4.5 × 106 cells per T175 cell culture flask one day prior to the assay. Next, cells were harvested by a enzyme-free cell dissociation solution (Sigma-Aldrich, St.Louis, USA). Cells were pelleted at 300 g for 5 min and washed twice with PBS. Cells were lysed with 200 µl RIPA lysis buffer containing a EDTA-free protease inhibitor (Sigma-Aldrich, St.Louis, USA) and a PhosStop phosphatase inhibitor (Sigma-Aldrich, St.Louis, USA) for 30 min on ice. After centrifugation at 10,800 rpm for 5 min, the supernatant was transferred to new tubes and the protein concentration was measured by a colorimetric DC protein assay (Bio Rad, Hercules, USA).
4
CD108 Expression in HLF and NIH3T3 Cells
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After the indicated culture conditions and treatments, HLF or NIH3T3 cells were detached using enzyme-free cell dissociation solution (Sigma-Aldrich, St. Louis, MO) supplemented with 10 mM EDTA. Cells were then stained with either PE-conjugated anti-CD108 (semaphorin-7A) or an isotype control (BD Biosciences, San Jose, CA). Flow histograms were generated using FlowJo analysis software (FlowJo, Ashland, OR).
5
Quantifying TLR2 Expression in Caco-2 Cells
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Caco-2 cells were detached using an enzyme-free cell dissociation solution (Sigma-Aldrich) to prevent the denaturation of surface proteins. After detachment, the cells were washed twice with cold PBS. The cells were stained with APC-conjugated anti-human TLR2 antibody and its isotype control for 30 min on ice and then washed twice with PBS. The stained cells were fixed using 1% paraformaldehyde, and the expression of TLR2 on the cell was analyzed using flow cytometry (FACSVerse, BD Biosciences).
6
Viability and Adherence Capacity of Irradiated CAFs
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CAFs from the same donors as those used in the animal experiments were irradiated in the same way as cells utilized for in vivo studies, and 24 hours post-irradiation examined for viability and adherence capacities. For that purpose, twenty-four hours after the last radiation exposure, cells were incubated with Enzyme-Free Cell Dissociation Solution (Merck Millipore), then detached and spun down (400 g, 3 min). Resulting cell pellets were dissolved in 500 μL cold medium and kept on ice for 45 min to mimic the procedure for cell implantation. Cell viability was determined by Trypan blue exclusion assay, and percentage viable cells was estimated by dividing number of cells without dye against total number of cells. For analysis of plating efficiency, the harvested cells were transferred to a T-25 flask and incubated further (24 hours, 37 °C) for cell adherence. After incubation, non-adherent cells was counted, and plating efficiency (%) was calculated in relation to total sum of adherent and non-adherent cells.
7
EGFR Expression Analysis in Breast Cell Lines
8
Quantifying C3b Deposition on iGEnCs
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iGEnCs were seeded on a 24-well plate until they reached full confluency, and incubated with serum-free medium overnight. Staining for C3b was performed as described in [4], with a few changes. Cells were incubated for 30 min at 37°C with 10% C9-depleted NHS and 150 µg/ml of CRP, supplemented with a physiological concentration of a particular C2 variant. Detection of C3b and CD31 was conducted with anti-C3c-FITC (Dako)/CD31-PE antibodies (Sigma-Aldrich) diluted 1:200 in 0.1% BSA in PBS for 30 min in 4°C. Cells were washed with PBS and gently detached with enzyme-free cell dissociation solution (Merck Millipore) to be analyzed by flow cytometry using CytoFLEX (Beckman Coulter) or left on the plate and overlaid with a mounting medium with TRITC-Phalloidin (Vectashield) for fluorescent microscopy imaging (Olympus IX83).
9
Measuring BAFF Binding to TNFR Superfamily Receptors
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BAFF was biotinylated using Pierce NHS-LC-biotin (Thermo Fisher Scientific); HEK293 cells were stably transfected with human BR3, human TACI, or human BCMA. Expression was confirmed with specific antibody binding and flow cytometry. The cells were removed from the cell culture flask using Enzyme Free Cell Dissociation solution (EMD Millipore), resuspended at 106 cells/mL in FACS buffer (D-PBS, 2% FBS, 0.1% sodium azide) with 1 mg/mL goat IgG added. For cells expressing BCMA, 300 ng/mL biotinylated BAFF + 50 μg/mL tabalumab were pre-incubated for 15 minutes at RT. For BR3, TACI, or vector control cells, 18.8 ng/mL biotinylated BAFF + 5 μg/mL tabalumab were pre-incubated for 15 minutes at RT. The mixture was added to cells and incubated for 20 minutes on ice. The cells were washed with FACS buffer and resuspended with streptavidin-PE (phycoerythrin; Jackson ImmunoResearch) at a 1:150 dilution and incubated on ice for 15 minutes. The cells were washed as above and then resuspended in FACS buffer. The fluorescence was measured using a Guava EasyCyte Plus instrument (EMD Millipore) counting 2,000 cells with the Express Plus protocol. The files were converted to FCS files and the overlays were generated using WinList 5.0 (Verity Software House, Topsham, ME, USA).
10
Glucose-Induced Cell Viability Assay
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Effects of glucose stimulation on cell viability were determined using the VivaFix Cell Viability Assay (Bio-Rad Laboratories, Inc., Hercules, CA) that relies on fluorescent VivaFix dye that is concentrated into dead cells while excluded from live ones. INS-1E cells were seeded at an initial density of 5.0x105 cells/well in a 24-well plate. RPMI 1640 media was exchanged 24 h after cell seeding and experiments were conducted the following day. We stimulated cells with 20 mM glucose (90 min, 37°C) while unstimulated control cells remained in KRB buffer (0 mM glucose, 90 min, 37°C) according to conditions identical to our HTRF insulin assay. Cells from each respective condition were collected with Enzyme Free Cell Dissociation Solution (EMD Millipore, Billerica, MA) and resuspended in Dulbecco’s PBS buffer (DPBS; Thermo Fisher Scientific Inc., Waltham, MA). Cells were incubated with VivaFix dye (30 min, 37°C), washed twice with DPBS, and placed into a flow cytometer (BD Accuri C6 Flow Cytometer; BD Biosciences, Franklin Lakes, NJ). Labeled and unlabeled cells in both stimulated versus unstimulated conditions were then counted by plotting the differences in fluorescence intensity measured at 675 ± 12 nm using BD CSampler Software (BD Biosciences).
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