The secretion of elastin by fibroblasts was determined using a Fastin™ Elastin Kit (Biocolor, Northern Ireland, UK). Cells were seeded in a 12-well plate with 100,000 cells/well density and maintained in different experimental settings similar to the proliferation and migration assay. Cells were incubated at 37 °C in 5% CO2 for 48 h before processing according to the manufacturer’s instructions. Briefly, cells were released using CTSTM TrypLETM Select Enzyme (Thermo Scientific, MA, USA) and maintained in 300 μL of supernatant after centrifugation. Subsequently, an equal volume of Elastin Precipitating Reagent was added to each sample tube. The supernatant was discarded after centrifugation, and the pellets were resuspended in 1 mL Dye Reagent and incubated for 90 min. Pellets were then collected and resuspended in 250 μL of Dye Dissociation Reagent. A volume of 100 μL of suspension was transferred to a 96-well plate for reading OD at 513 nm using spectrometer SpectraMax M3 (Molecular Devices, CA, USA).
Ctstm tryple select enzyme
Manufactured by Thermo Fisher Scientific
Sourced in United States
CTSTM TrypLETM Select Enzyme is a cell dissociation reagent used in cell culture applications. It is a trypsin-like protease that can be used to detach adherent cells from culture surfaces. The product is formulated to provide efficient cell dissociation while maintaining cell viability and functionality.
Automatically generated - may contain errors
Lab products found in correlation
Spectramax m3, by Molecular Devices (1 mentions)
Fastin elastin kit, by Biocolor (1 mentions)
Human msc analysis kit, by BD (1 mentions)
Foetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Ctstm cellstart substrate, by Thermo Fisher Scientific (1 mentions)
Recombinant human bmp 2, by R&D Systems (1 mentions)
Unicel dxi 800 access immunoassay system, by Beckman Coulter (1 mentions)
Bm hmscs, by RoosterBio (1 mentions)
Mycoalert mycoplasma detection kit, by Lonza (1 mentions)
Roosternourish msc xf, by RoosterBio (1 mentions)
5 protocols using ctstm tryple select enzyme
1
Quantifying Fibroblast Elastin Secretion
2
MSC Marker Analysis Protocol
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The UCMSCs at P5 were harvested following supernatant collection using CTS TM TrypLe TM Select Enzyme (Thermo Fisher Scientific, USA). Then they were subjected to marker analysis using Human MSC Analysis Kit (BD Biosciences, California, USA). The kit includes the MSC positive cocktail (FITC CD90, PerCP-CyTM5.5 CD105, and APC CD73) and the MSC negative cocktail (PE: CD45, CD34, CD11b, CD19, and HLA-DR). Staining was performed according to the manufacturer's instructions. Flow cytometry was performed using a Beckman Coulter flow cytometer equipped with Navious software.
3
Expansion and EV Isolation from UCMSCs
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UCMSCs were received from the EV groups at passage two (P2). The cells were thawed in the water bath (37 o C) and seeded into T75, or T225 cell culture flasks (Nunc, Thermo Scientific, Massachusetts, United States) containing DMEM/F12 (Dulbecco's Modified Eagle's Medium, Gibco, USA) supplemented with 10% FBS (Foetal Bovine Serum, Gibco, USA) with a density of 375 × 10 3 cells/cm 2 as passage 3. The culture flask was surface-coated with CTS TM CELLstart TM substrate (Gibco, Massachusetts, USA) diluted in PBS at the rate of 1: 300 and washed twice with PBS before cell seeding. UCMSCs were then incubated at the condition of 5% CO 2 /37 o C for the expansion. When cells reach 80% confluency, they were split using CTS TM TrypLE TM Select Enzyme (Thermo Fisher Scientific, USA) for the next passage. At the culture of P5, UCMSCs were incubated to reach 80% confluency and released EVs into conditioned media (supernatant) that were harvested for further EV isolation. The UCMSCs were split for marker analysis.
4
Investigating BMP-2 Effects on H295R Steroidogenesis
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H295R cells were cultured on six-well plates precoated with an extracellular matrix at a density of 18 × 104 cells per well and cultured for 48 h. Cells were treated with 0 to 50 ng/mL recombinant human BMP-2 (R & D Systems, Minneapolis, MN, USA) in H295R culture media for 48 h. These concentrations were chosen based on a previous study using bovine theca cells [69 (link)]. After removal of treatment media, the cells were washed with PBS three times before adding basal media (serum-free) and incubating for another 24 h. To compare the cell number, cells was determined using a CTSTM TrypLE select enzyme (Gibco, Waltham, MA, USA). The number of live cells was counted by Trypan blue assay, which is the most widely used and still the gold standard method to perform cell viability assays in cell culture [70 (link),71 (link)]. Cells were collected to analyze the expression of steroidogenesis pathway genes. The cell culture supernatant was used for chemiluminescent quantification of testosterone released by H295R cells using an automated UniCel DxI 800 Access Immunoassay System (Beckman Coulter, Inc., Brea, CA, USA) [72 (link)].
5
Bone Marrow-Derived Mesenchymal Stem Cell Expansion
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BM-hMSCs were purchased from Roosterbio (Frederick, MD, USA). These cells were originally isolated from the bone marrow of a 29-year-old female donor. For BM-hMSCs culture, around 3000 cells/cm2 were plated in a Corning CellBIND® T75 cell culture flask (Corning, NY, USA). BM-hMSCs were cultured with the recommended cell culture medium, RoosterNourish™-MSC-XF (Roosterbio, Frederick, MD, USA), per the expansion protocol. When the culture reached approximately 80% confluence, cells were trypsinized using CTSTM TrypLE select enzyme (Gibco, Waltham, MA, USA) and serially expanded for two more passages before use in experiments. Parallel flasks were used for the collection of conditioned media (BM-hMSCs secretome). All cultured cells in this study were tested for mycoplasma using a MycoAlertTM mycoplasma detection kit (Lonza, Basel, Switzerland), and all cells cultured were free from mycoplasma contamination.
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