As previously described (Lalancette-Hebert et al., 2012 (link); Rahimian et al., 2019b (link)), the primary cell culture was performed on the brains of the adult, 8–9 weeks old C57BL/6 wild-type mice. The mice were anesthetized by isoflurane 2% and transcardially perfused with ice-cold saline (Hospira) supplemented with 2 units/ml heparine (Sigma-Aldrich). The brains were collected and placed in ice-cold Hibernate medium [Hibernate A medium (BrainBits LLC) supplemented with B-27 (1X) and 0.5 mM GLUTAMAX-I, L-alanyl-L-glutamine (Gibco, Invitrogen)]. After mechanical dissociation, 5–6 brains were incubated in a 0.25% Trypsin-EDTA solution (Sigma) containing 250 K U/ml of DNase I (Sigma). After mechanical dissociation, 5–6 brains were incubated in 0.25% Trypsin-EDTA solution (Sigma) containing 250 K U/ml of DNase I (Sigma). After centrifugation, the pellet was resuspended in 2 ml of 37% Percoll that was overlaid 2 ml of 70% Percoll in a 5-ml centrifuge tube and centrifuged at 600×g for 40 min at room temperature with slow acceleration and no stop-brake. Cells were collected from the interphase, washed with dPBS and kept in culture medium. Cells were plated on culture-treated glass slides (Becton Dickinson) as 1,250,000 cell/ml and incubated at 37 °C in 95% air/5% CO2 (Lalancette-Hebert et al., 2012 (link); Rahimian et al., 2019b (link)).
Trypsin edta solution
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Trypsin-EDTA solution is a cell culture reagent used to detach adherent cells from their growth surface. It contains the proteolytic enzyme trypsin and the chelating agent EDTA, which together disrupt the cell-to-cell and cell-to-substrate adhesions, allowing cells to be harvested and subcultured.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (79 mentions)
Fetal bovine serum (fbs), by Merck Group (63 mentions)
Penicillin streptomycin, by Merck Group (56 mentions)
L glutamine, by Merck Group (42 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (38 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (34 mentions)
Streptomycin, by Merck Group (33 mentions)
Penicillin, by Merck Group (32 mentions)
Dulbecco s modified eagle s medium dmem, by Merck Group (30 mentions)
Penicillin streptomycin solution, by Merck Group (28 mentions)
Phosphate buffered saline (pbs), by Merck Group (27 mentions)
Streptomycin, by Thermo Fisher Scientific (26 mentions)
Penicillin, by Thermo Fisher Scientific (25 mentions)
Rpmi 1640 medium, by Merck Group (23 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (22 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (21 mentions)
Penicillin streptomycin solution, by Thermo Fisher Scientific (17 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (14 mentions)
3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide mtt, by Merck Group (14 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (12 mentions)
605 protocols using trypsin edta solution
1
Primary Culture of Mouse Brain Cells
2
Primary and Immortalized Astrocyte Culture
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For primary astrocytes isolation and culture, primary astrocytes were dissected from newborn Mysm1fl/fl or C57BL/6N mice. The brain cortex and hippocampus were dissected, and the meninges were removed under a microscope. Then, the tissue was dissociated by enzymatic digestion, and the isolated astrocytes were cultured in astrocytic medium (Dulbecco's Modified Eagle's Medium/Nutrient mixture F‐12 Ham) (DMEM/F12) (1:1) (Sigma, D8437) with 10% fetal bovine serum (FBS; ExCell Bio, 12B013) and antibiotics (100 U mL−1 penicillin and 100 µg mL−1 streptomycin, Gibco) in a dish plated with poly‐D‐lysine (Sigma, P6407) and cultured in a 5% CO2 incubator at 37 °C. Astrocytes were grown for ≈7 days until the cultures reached the desired density, with medium changes every 2 days. Trypsin‐EDTA solution (0.25%, Sigma) was used to dissociate primary astrocytes. Cells from passage 2–3 were used for the experiments.
For C8‐D1A astrocytes, cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco, Thermo Fisher Scientific, D6429) with high glucose and supplemented with 10% FBS and antibiotics at 37 °C in humidified 5% CO2 incubator. Trypsin‐EDTA solution (0.25%, Sigma) was used to dissociate C8‐D1A cells. Cells from passage 2–15 were used for the experiments.
For C8‐D1A astrocytes, cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco, Thermo Fisher Scientific, D6429) with high glucose and supplemented with 10% FBS and antibiotics at 37 °C in humidified 5% CO2 incubator. Trypsin‐EDTA solution (0.25%, Sigma) was used to dissociate C8‐D1A cells. Cells from passage 2–15 were used for the experiments.
3
Evaluating RPC Proliferation, Adhesion, and Spreading
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For cell proliferation experiments, RPCs cultured in 1 × g and modeled μg conditions for 72 h were detached using a 0.25% trypsin–EDTA solution (Sigma-Aldrich, St. Louis, MO, USA) and then counted.
For cell adhesion and cell spreading analysis, RPCs cultured in 1 × g and modeled μg were detached from the beads using 0.25% trypsin–EDTA solution (Sigma-Aldrich, St. Louis, MO, USA) and plated into multiwell plates in EGM-MV + 20% FBS at a density of 8 × 103 cells/cm2. Cell analysis was conducted at 1, 3 or 24 h by using the EVOS XL Cell Imaging System (Thermo Fisher Scientific). To analyze cell adhesion through crystal violet staining, cells were plated as described above. After 3 h, cells were fixed for 15 min in 4% paraformaldehyde, stained with 0.1% (w/v) crystal violet for 20 min at room temperature, and then washed with Dulbecco’s Phosphate Buffer Saline (D-PBS). The stained cells were dissolved in 2% SDS for 30 min, and the absorbance was measured at 560 nm using the FLUOstar Optima Microplate Reader (BMG LABTECH, Ortenberg, Germany).
For cell adhesion and cell spreading analysis, RPCs cultured in 1 × g and modeled μg were detached from the beads using 0.25% trypsin–EDTA solution (Sigma-Aldrich, St. Louis, MO, USA) and plated into multiwell plates in EGM-MV + 20% FBS at a density of 8 × 103 cells/cm2. Cell analysis was conducted at 1, 3 or 24 h by using the EVOS XL Cell Imaging System (Thermo Fisher Scientific). To analyze cell adhesion through crystal violet staining, cells were plated as described above. After 3 h, cells were fixed for 15 min in 4% paraformaldehyde, stained with 0.1% (w/v) crystal violet for 20 min at room temperature, and then washed with Dulbecco’s Phosphate Buffer Saline (D-PBS). The stained cells were dissolved in 2% SDS for 30 min, and the absorbance was measured at 560 nm using the FLUOstar Optima Microplate Reader (BMG LABTECH, Ortenberg, Germany).
4
Culturing and Differentiating PC-12 and 3T3 Cells
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A PC-12 (ATCC® CRL-1721™) cell line (P3) was cultured in RPMI-1640 media (Sigma-Aldrich) supplemented with 10% horse serum (Hyclone), 5% FBS (Hyclone) and 1% penicillin-streptomycin solution (Sigma-Aldrich). Cells were incubated at 37°C in 5% CO2.
Cells were passaged weekly using a 0.25% trypsin-EDTA solution (Sigma). The growth medium was changed to differentiating medium containing RPMI-1640 supplemented with 1% horse serum, 1% penicillin-streptomycin solution and 100 ng/mL nerve growth factor (NGF, Sigma-Aldrich) for neuronal differentiation after 24 h of culture.
A mouse embryonic 3T3 fibroblast cell line (P15), NIH 3T3 (ATCC® CRL-1658TM) was used to investigate the impact of the electrospun meshes on cell viability. Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS and 1% penicillin-streptomycin (Pen Strep) and were incubated at 37°C in 5% CO 2 . Cells were passaged weekly using a 0.25% trypsin-EDTA solution (Sigma).
Cells were passaged weekly using a 0.25% trypsin-EDTA solution (Sigma). The growth medium was changed to differentiating medium containing RPMI-1640 supplemented with 1% horse serum, 1% penicillin-streptomycin solution and 100 ng/mL nerve growth factor (NGF, Sigma-Aldrich) for neuronal differentiation after 24 h of culture.
A mouse embryonic 3T3 fibroblast cell line (P15), NIH 3T3 (ATCC® CRL-1658TM) was used to investigate the impact of the electrospun meshes on cell viability. Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS and 1% penicillin-streptomycin (Pen Strep) and were incubated at 37°C in 5% CO 2 . Cells were passaged weekly using a 0.25% trypsin-EDTA solution (Sigma).
5
Isolation and Characterization of Human Amniotic Epithelial Cells
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hAEC were isolated from term placentas (n = 5) as previously described 24 . Briefly, the amnion was detached from chorion and washed in PBS (Sigma‐Aldrich, St Louis, MO, USA) supplemented with 100 U/ml penicillin (Lonza, Basel, Switzerland) and 100 μg/ml streptomycin (Sigma‐Aldrich). Amnion fragments ( ~15 × 15 cm) were incubated for 10 min. at 37°C in PBS containing 0.5 mM EDTA and P/S, and then in 1X trypsin/EDTA solution (Sigma‐Aldrich) for 5 min. at 37°C. After discarding debris, the fragments were again incubated (10 min. at 37°C) in fresh trypsin/EDTA solution and, after washing in PBS, were once more digested in trypsin/EDTA. The cells from the second and third digestions were pooled and centrifuged at 300× g for 10 min. Cell suspensions were then filtered through a 70‐μm cell strainer (BD Biosciences, San Jose, CA, USA), centrifuged and counted. Isolated cells were cryopreserved in 10% DMSO (Sigma‐Aldrich) supplemented with 90% FBS until use.
hAEC batches with cells over 95% positive for CD324 and CD166, and negative for CD44, CD105 and CD45 were used. Cell viability after thawing was always higher than 85%.
hAEC batches with cells over 95% positive for CD324 and CD166, and negative for CD44, CD105 and CD45 were used. Cell viability after thawing was always higher than 85%.
6
Candida Strains and Cell Line Maintenance
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Candida strains (listed in S1 Table ) were provided by the Dr. P. David Rogers lab of the University of Tennessee Health Science Center, Memphis, TN. Human cell lines; THP-1 (ATCC, TIB-202), HepG2 (ATCC, HB-8065), and A549 (ATCC, CCL-185) were purchased from the American Type Culture Collection (Manassas, VA, USA). Dulbecco′s Modified Eagle′s culture medium (DMEM), 1X trypsin-EDTA solution, fetal bovine serum (FBS), 100X penicillin/streptomycin solution, Amphotericin B, caspofungin, fluconazole, itraconazole, 5-fluorocytosine, YPD agar and broth, cytochalasin D, 3-(N-morpholino) propanesulfonic acid (MOPS) buffer, and methyl methanesulfonate (MMS) were all purchased form Sigma-Aldrich (St. Louis, MO, USA). RPMI-1640 medium was purchased from Corning Incorporated (Coring, NY, USA). Geneticin selective antibiotic (G-148 sulfate), phosphate buffered saline (PBS), rhodamine phalloidin, propidium iodide, PrestoBlue, and RNaseA enzyme were purchased from Life Technologies Corporation (Carlsbad, CA, USA). 4% Paraformaldehyde was purchased from Alfa Aesar (Ward Hill, MA, USA) and zymolase 20T was purchased from MP Biomedicals, LLC (Solon, OH, USA). The yeast deletion collections (~1,056 heterozygous mutants and ~4,320 homozygous mutants) were purchased from GE Healthcare Life Sciences (Pittsburg, PA, USA) and ThermoFisher Scientific (Waltham, MA, USA), respectively.
7
Culturing and Characterizing A549 Lung Cells
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Authentic A549 cells (European Collection of Cell Cultures (ECACC), Salisbury, UK), catalogue number 86012804, were cultured in either Ham’s F12 Nutrient Medium (Ham’s F12) or Dulbecco’s Modified Eagles Medium (DMEM) (both from Sigma Aldrich, Dorset, United Kingdom) supplemented with 2mM L-Glutamine and 10% v/v Foetal Bovine Serum (FBS) (Hyclone SH30071.03 (Hyclone Laboratories, Utah, USA). Proliferative cultures were incubated at 37°C in a humidified 5% CO2 incubator and subculture carried out by washing the cell monolayers twice with calcium and magnesium-free phosphate buffered saline (PBS) (Severn Biotech (Kidderminster, UK, catalogue number 20–74) followed by addition of 1x Trypsin/EDTA solution (Sigma Aldrich) and incubation at 37°C until the cells detached. Trypsin was inactivated by the addition of growth medium before seeding into fresh flasks at densities of 1.5-2x104 cells/cm2. For the long term 25 day cultures A549 cells were seeded into replicate T25 flasks and medium changed every 2–4 days. Phase contrast images were captured of the monolayers throughout the time course. Cell numbers, viability and size were assessed by Trypan Blue staining and by DAPI dye exclusion using the Nucleocounter™ 3000 viability assay (Chemometec, Allerod, Denmark).
8
Cell Cycle Analysis by Flow Cytometry
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HEK293 cells, HeLa cells, or HH-12 chicken embryos were electroporated with pSHIN-shControl or pSHIN-shVCL plasmids for 24 h. A single-cell suspension was obtained by treating each 10-cm cell dish or 10 embryos (HH18) with 1 ml of 1X Trypsin-EDTA solution (Sigma-Aldrich) containing 100 U of DNAseI for 5–10 min at 37°C. Digestion was stopped by adding one volume of culture media containing 10% of FBS. Individual cells were collected by centrifugation at 300 g for 5 min. Cells were resuspended in 300 μl of PBS and fixed for 15 min adding 1 ml of 4% PFA. PFA was blocked by adding 1 vol of PBS containing 10% of FBS and 0.1% Triton X-100. Cells were centrifugated at 300 g for 5 min and then resuspended in PBS-BSA containing Hoechst and RNase A. Hoechst and GFP fluorescence were determined by FACSAria Fusion cytometer (BD Biosciences) and the data were analyzed with FlowJo software (Tree Star) and Multicycle software (Phoenix Flow Systems; cell cycle profile analysis).
9
Isolation of Porcine Corneal Endothelial Cells
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Corneal endothelial cells were isolated from freshly enucleated porcine eyes using a trypsin/EDTA digestion protocol first described by Xie and Gebhardt.34 (link) Briefly, after the porcine eyes were dissected and their intact corneas isolated from the globe, 30-μL 10X trypsin/EDTA solution (Sigma-Aldrich Corp., St. Louis, MO, USA) was applied directly to the endothelial surface of the cornea to disengage CECs from Descemet's membrane. Corneas were then incubated with 10X trypsin/EDTA for 5 minutes at 37°C. Corneal endothelial cells removed from Descemet's membrane were subsequently washed and collected using a 200-μL pipette tip before being directly transferred to a sterile, 60 × 15-mm Falcon petri dish coated with 10 μg/mL mouse collagen IV (Trevigen, Gaithersburg, MD, USA) and resuspended in growth media. Growth media consisted of low glucose Dulbecco's modified Eagle's medium (Gibco BRL Life Technologies, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/mL), streptomycin (100 mg/μL), gentamicin (15 μg/mL), and 75 μg/mL endothelial cell growth supplement from bovine pituitary (Sigma-Aldrich Corp.). Corneal endothelial cells were incubated at 37°C until confluence was achieved (∼12 days). Growth media used in primary culture and serial passages was exchanged every 72 hours.
10
Dissecting and Analyzing Larval Wing Discs
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Approximately 20 third instar larval wing discs of each genotype were dissected in PBS and transferred to 0.5 mL of 10x Trypsin-EDTA solution (Sigma-Aldrich) and incubated for 3 hours at room temperature on a nutator. The cells were analyzed using FACScalibur and BD CellQuest Pro.
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