The cells were trypsinized [Trypsin EDTA Solution B (0.25%), EDTA (0.05%)] (Biological industries, Beit Ha’Emek, Israel) before seeding into 24-well plate. The procedure was performed by the mixture of cells and trypan blue dye (1:1) in an Eppendorf tube. Ten microliters of this mixture were used on the specific Neubauer’s slide and covered by cover slip. The cells were counted manually by the hemocytometer and the number of the cells was recorded and calculated per milliliter.
Trypsin edta solution b
Manufactured by Sartorius
Sourced in Israel, United States
Trypsin EDTA Solution B is a laboratory reagent used for cell detachment and dissociation. It contains the proteolytic enzyme trypsin and the chelating agent EDTA, which work together to break down the adhesive proteins that hold cells to a surface or to each other. This solution is commonly used in cell culture procedures to release adherent cells from the growth surface, enabling their subsequent passage or collection.
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Lab products found in correlation
Trypsin inhibitor, by Merck Group (2 mentions)
Trypsin edta solution b, by Merck Group (2 mentions)
Propidium iodide, by Merck Group (2 mentions)
Minimum essential medium (mem), by Sartorius (1 mentions)
Actinomycin d a 1410, by Merck Group (1 mentions)
Polylysine adhesion microscope slide, by Electron Microscopy Sciences (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Phosphate buffered saline (pbs), by Sartorius (1 mentions)
Human fibronectin, by Merck Group (1 mentions)
Huvecs, by Lonza (1 mentions)
Endothelial cell medium (ecm), by ScienCell (1 mentions)
Fluorescein diacetate, by Merck Group (1 mentions)
Live cell imaging solution, by Thermo Fisher Scientific (1 mentions)
Live dead cell imaging kit, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin solution, by Sartorius (1 mentions)
Sodium pyruvate solution, by Sartorius (1 mentions)
L glutamine, by Sartorius (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Glacial acetic acid, by Carlo Erba (1 mentions)
Synergy h1, by Agilent Technologies (1 mentions)
12 protocols using trypsin edta solution b
1
Cell Counting using Trypan Blue
2
Vitamin D Signaling Regulation
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MEM, fetal calf serum (FCS), L-glutamine, antibiotics mixture (penstrepnystatin) and trypsin-EDTA solution B were purchased from Biological Industries. Tissue culture dishes were purchased from Corning Glass Work. 1α,25-dihydroxyvitamin D3 (calcitriol) was obtained from Teva Pharmaceutical Industries Ltd. BSA fraction V (160069) was purchased from ICN Biomedicals, Inc.. BCA Protein Assay Kit (23225) was obtained from Pierce Biotechnology Inc.. Human recombinant tumor necrosis factor α (TNFα) (300-01A), interleukin-1β (IL-1β) (200-01B) and interferon γ (IFNγ) (300-02L) were obtained from PeproTech Inc.. U0126 (BML-EI282-0001) and SB203580 (BML-EI286-0001) were purchased from Alexis Biochemicals. SP600125 (420119) and BMS-345541 (401480) were purchased from Calbiochem. Actinomycin D (A-1410) was purchased from Sigma Chemical Co. Mouse monoclonal anti-VDR (D-6) (sc-13133) and polyclonal rabbit anti RXRα (sc-553) were obtained from Santa Cruz Biotechnology. IRDye 800CW goat anti-mouse IgG (925-32210) and IRDye 800CW goat anti-rabbit IgG Abs (926-32211) were from LICOR. All other reagents are of analytical grade.
3
Huh-7.5 Cell HCV Infection and Characterization
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Growth and infection of Huh-7.5 cells with HCV were carried out as described in24 (link). Briefly, cells were infected with the HJ3-5 chimeric virus at an MOI of 0.1–0.5 and passaged for two weeks until approximately 100% of the cells were HCV positive, as previously described24 (link). High infection levels were maintained for two weeks by adding 2% human serum to the culture medium, as previously described. HCV-infected and uninfected cells were washed twice with trypsin EDTA Solution B (Biological Industries), spread onto a polylysine adhesion microscope slide (Electron Microscopy Sciences), and incubated for two hours at 37 °C. The cells were fixed in methyl alcohol/acetone for 10 min at room temperature. The slides were then stained with mounting medium, including DAPI (Sigma-Aldrich), and covered with a coverslip (Kaltek).
4
FTIR Analysis of Cell Samples
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The cells were washed 1 × with PBS (02-023-1A Biological Industries, Beit Haemek, Israel) and trypsinised using Trypsin EDTA solution B (03-052-1B Biological Industries, Beit Haemek, Israel), and centrifuged at 400 g for 5 min at 4 °C and re-suspended in normal growth medium to the appropriate concentration as indicated in the experiments. 10 µl of sample was subsequently dropped on the diamond ATR in the FTIR instrument for acquiring absorbance spectra.
5
HUVEC Culture and Perfusion in a Microdevice
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Human umbilical vein endothelial cells (HUVECs, Lonza) were maintained in endothelial cell medium (ECM, ScienceCell, Carlsbad, CA, USA). Cells were cultured at 37 °C, in a humidified environment, with 5% CO2. Cells were passaged every 4–5 days, i.e., when they reached 85% confluence, using Trypsin EDTA solution B (Biological Industries, Cromwell, CT, USA). The microdevice was coated with human fibronectin (100 µg/mL, Sigma), overnight, at 4 °C. HUVECs at passages 4–6 were seeded in the microdevice at a density of 1.5 × 106 cells/mL and incubated for two hours to allow the cells to adhere. The microdevice was connected to a programmable syringe pump for controlled perfusion overnight (flow rate 50 µL/h). Figure 1 b shows a phase microscopy image of ECs grown under perfusion within the device.
6
Degradation of Structural Maintenance of Chromosomes Protein
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Degradation of SMC was tested using an acidic solution (pH 1.8) and the trypsin/EDTA Solution B (0.25% trypsin and 0.05% EDTA in Puck's Saline A) from Biological Industries – Israel. We dissolved 1 mg SMC in 1 ml double distilled water (SDDW) containing 2 μl hydrochloric acid (final pH = 1.8) or in 1 ml of the trypsin-containing solution described above – modified from (Thiede et al., 2000 (link)). HPLC analysis (described below) was conducted for evaluating the obtained results after degradation.
7
Alginate Hydrogel Preparation and Characterization
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l -glutamine, penicillin/streptomycin solution, trypsin-EDTA solution B, and sodium pyruvate solution were purchased from Biological Industries (BI), Beit HaEmek, Israel. Fetal bovine serum (FBS) was from Gibco, Life Technology, Grand Island, NY, USA. Alginate (A2033, medium MW 3.5 × 105 g/mol, M/G ratio: 1.56 [61 (link)]), propidium iodide (PI), fluorescein diacetate (FDA), HEPES Sodium 2-acrylamido 2-methyl propane sulfonic acid (NaAMPS) (58% in aqueous solution), polyethylene glycol diacrylate (PEG575DA, Mn ~500), ascorbic acid (AA), potassium peroxymonosulfate (Oxone), polyethylene glycol diacrylate (PEGDA), and 3-Sulfopropyl acrylate potassium salt (KSPA) were purchased from Sigma, Rehovot, Israel. Acetic acid glacial was from Carlo ERBA, Val-de-Reuil, France. Calcium carbonate (CaCO3) was purchased from DAEJUNG, Siheung-si, Gyeonggi-Do, Korea. Dil (1,1′-dioctadecyl-3,3,3′3′-tetramethylindocarbocyanine perchlorate) Cell-labeling solution and Live/Dead™ Cell Imaging kit were purchased from Invitrogen™, Waltham, MA, USA. Live Cell Imaging Solution was from Molecular Probes™, Eugene, OR, USA.
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8
Isolation of Primary Mouse Myoblasts
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Primary mouse myoblasts were isolated from gastrocnemius muscle using mechanical tissue dissociation as in ref. Eigler et al, 2021 (link). Briefly, after cutting the muscle tissue into small pieces, they were incubated in Trypsin EDTA Solution B (0.25% Trypsin and 0.5% EDTA, Biological Industries Israel) and subjected to mechanical dissociation with a serological pipet. Supernatants were strained (FALCON REF no. 352340) and centrifuged. Cell pellets were resuspended in BioAMFTM-2 media (Biological Industries, Israel), plated on 10% Matrigel® Matrix (Corning REF no. 354234) coated plates, and grown at 37° in a 5% CO2 incubator. BioAMFTM-2 was used in all experiments (Biological Industries Israel).
9
Cytokine Profiling and Apoptosis Analysis
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Cytokine levels in sampled medium supernatants were measured using commercially available ELISA kits in a blinded fashion and by following the manufacturer's directions (Invitrogen) for the following cytokines: IL‐6 (#88–7066‐77), IL‐8 (#88–8086‐77), and IL‐10 (#88–7106‐88). For flow cytometry, cells were dissociated from the lumen of the model by adding Trypsin EDTA Solution B (0.25%), EDTA (0.05%) (Biological Industries). Following collection, culture medium (see culture) was added at a 1:1 ratio to block the Trypsin‐B effect. Cells were subsequently centrifuged at 3.0 rpm for 3 min., the supernatant discarded and then resuspended in 500 μl of binding buffer, stained with 10 μl of AnnexinV‐FITC and PI following manufacturer's instructions (Annexin‐V Apoptosis Staining Detection Kit, ab14084, Abcam). Metabolic activity tests were performed using an alamarBlue cell viability assay (BUF012, Bio‐rad). The absorbance of the incubated media containing 10% alamarBlue (2 h) was measured at 570 and 600 nm using a microplate reader (Synergy H1, BioTek).
10
Cellular Spreading on Extracellular Matrix
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To study the cell spreading on fibronectin and galectin-8-coated substrate, cells from 70–80% confluent cultures in 22.1 mm wells of multi-well dishes were first washed with warm PBS once, then incubated in 150 µl of trypsin-EDTA solution B (Trypsin 0.25%, EDTA 0.05%) (Biological Industries Cromwell, CT, catalog no. 03-052-1B) at 37°C for 2 min and gently suspended by addition of 5 ml serum-free DMEM with trypsin inhibitor (T9003, Sigma-Aldrich) (1 mg of trypsin inhibitor per ml trypsin-EDTA solution B). The cell suspension was centrifuged at 1000 rpm for 5 min, supernatant was removed and serum-free medium was added to re-suspend the cells. Then, cells were plated onto pre-coated Petri dishes and either imaged or fixed at appropriate time points.
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