The selected UC-MSC lines at P4 were thawed in prewarm StemMACS™ MSC expansion medium (Miltenyi Biotec) and centrifuged at 500×g for 5 min at room temperature. The cells were then resuspended in StemMACS medium and reseeded into a T75 Nunc™ EasYFlask™ Cell Culture Flask at a density of 3200 cells/cm2. Once the culture reached 80% confluence, the cells were incubated with TrypLE Select for 4 min at 37 °C and diluted with StemMACS medium, followed by centrifugation at 400×g for 5 min. The cells were counted and seeded into Corning® CellBIND® Surface HYPERFlask® cell culture vessels at a density of 3400 cells/cm2. When the culture reached 80% confluence, the cells were harvested for further analysis and cryopreserved in CryoStor CS10 cell freezing medium (Stemcell Technologies, Canada), followed by storage in liquid nitrogen for further use.
Cellbind surface hyperflask cell culture vessels
Manufactured by Corning
The CellBIND® Surface HYPERFlask® cell culture vessels are cell culture products designed to provide a large surface area for cell growth and expansion. These vessels feature a unique surface treatment that facilitates cell attachment and proliferation. They are intended for use in various cell culture applications.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Cryostor cs10 cell freezing medium, by STEMCELL (1 mentions)
Stemmacs msc expansion medium, by Miltenyi Biotec (1 mentions)
Human msc analysis kit, by BD (1 mentions)
Facslyric flow cytometer, by BD (1 mentions)
Histrap hp column, by GE Healthcare (1 mentions)
High glucose dmem, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
3 protocols using cellbind surface hyperflask cell culture vessels
1
Expansion and Cryopreservation of UC-MSCs
2
Comprehensive Characterization of UC-MSCs
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UC-MSCs harvested from either Corning® CellBIND® Surface HYPERFlask® cell culture vessels or the Quantum® Cell Expansion System were subjected to quality control tests, including tests for cell yield, cell viability (Trypan Blue), MSC markers and differentiation. The identification of MSCs included evaluations of the expression of positive MSC markers (CD73, CD90, and CD105) and negative markers (CD11b, CD19, CD34, CD45 and HLA-DR) using the Human MSC Analysis Kit (Becton Dickinson, USA). The cells were analyzed using a FACSLyric flow cytometer (Becton Dickinson, USA), followed by data analysis using FlowJo software. To assess differentiation ability, the harvested UC-MSCs were tested using a StemPro Osteogenesis, Adipogenesis, and Chondrogenesis Differentiation kit following the manufacturer’s instructions. Once differentiation was completed, the cells were fixed with 4% paraformaldehyde and stained with Alizarin Red S, Oil Red O, and Alcian Blue to detect osteogenic, adipogenic, and chondrogenic lineages, respectively.
3
Cell Surface Protein Labeling via Sortase
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Cell culture of L cells was performed in Corning® CellBIND® Surface HYPERFlask® cell culture vessels according to the manufacturer’s recommendations. L cells were grown in DMEM (high glucose) (Thermo Fisher Scientific) supplemented with 10% FBS (Thermo Fisher Scientific) and 2 mM L-glutamine (Thermo Fisher Scientific). In order to harvest the cells, cells were washed with 1 × PBS and trypsinized. The cell suspension was centrifuged (300 × g, 5 min, RT), pelleted cells were resuspended in 1× PBS and counted. Cells were then washed twice using 10 ml of tris (hydroxymethyl) aminomethane (Tris) buffer (50 mM Tris-HCl, 150 mM NaCl, 1.8 mM CaCl2, pH 7.5 at 37°C). His-tagged Sortase (Δ59SortA) (29 (link)) was expressed using the pET28(a)-SortA vector and purified using HisTrap HP columns (GE Healthcare). The final SortA concentration was 25 µM. 3G-Twin-Strep-tag (GGGWSHPQFEKGGGSGGGSGGSAWSHPQFEK, purity ≥ 95%) (peptides&elephants) was added in a molar ratio of 1:25 of SortA to tag. The calcium concentration was adjusted to 1.8 mM. The cells were incubated with SortA for 3 h at 37 °C while shaking at 600 rpm. After incubation, the cells were centrifuged (300 × g, 5 min, 4°C). The collected cells were kept for flow cytometric analysis, while the supernatant containing cleaved mCD1d was further purified.
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