Cell number was assessed at 1, 3 and 7 days of cell encapsulation through the detection of lactate dehydrogenase (LDH) activity of the cell lysate contained in the hydrogels. Briefly, hydrogels were washed with DPBS Ca2+ Mg2+ (Invitrogen) and incubated at 37 °C for 1 h in Eppendorf microtubes containing 50 μL of 3 mg/mL of collagenase type II (Invitrogen) for complete gel degradation. Thereafter, cells were lysed by adding 400 μL Mammalian Protein Extraction Reagent (M-PER; Thermo Fisher) followed by incubation for 30 min in ice. Samples were sonicated with an ultrasonic processor (UP50H, Hielscher) in cold to ensure complete cell lysis and centrifuged at 10,000 rpm for 10 min at 4 °C. Supernatant was used with the LDH quantification kit (Roche) following the manufacturer’s instructions and cell concentration was calculated through a calibration curve made with cell lysates of known cell concentration. Absorbance was read at 490 nm with an Infinite M200 PRO multimode plate reader instrument (Tecan). The experiment was carried out three times using triplicates per condition.
Dpbs ca2 mg2
Manufactured by Thermo Fisher Scientific
Sourced in United States
DPBS Ca2+ Mg2+ is a buffered saline solution that contains calcium and magnesium ions. It is commonly used in cell culture applications to maintain the ionic balance and osmolarity of the cell environment.
Automatically generated - may contain errors
Lab products found in correlation
Celltrace cfse dye, by Thermo Fisher Scientific (2 mentions)
Pre sort buffer, by BD (2 mentions)
Sh800s cell sorter, by Sony (2 mentions)
Collagenase type 2, by Thermo Fisher Scientific (1 mentions)
Ldh quantification kit, by Roche (1 mentions)
M per mammalian protein extraction reagent, by Thermo Fisher Scientific (1 mentions)
Infinite m200 pro, by Tecan (1 mentions)
Up50h, by Hielscher (1 mentions)
Senescence β galactosidase staining kit, by Cell Signaling Technology (1 mentions)
Crystal violet, by Merck Group (1 mentions)
35 mm dish, by BD (1 mentions)
5 protocols using dpbs ca2 mg2
1
Cell Viability Quantification in Hydrogels
2
Senescence-Associated β-Galactosidase Assay Protocol
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The senescence-associated β-galactosidase (SA-β-gal) assay was carried out using senescence β-galactosidase staining kit (Cell Signaling Technology, Danvers, MA, USA, http://www.cellsignal.com ) according to the manufacturer's instructions. Cells were cultured in a 35-mm plate and allowed to attach in growth medium until it reached 80% confluency. Cells were then washed twice with DPBS - Ca2+, - Mg2+ (Invitrogen), fixed and stained using one mL staining solution in citrate buffer (pH 6.0) overnight at 37°C. The developments of blue coloured cells were observed under a phase-contrast microscope using 20X magnifications. Five random fields were taken from each of the three replicate samples of ASCs (liposuction) and ASCs (biopsy) as well as BM-MSCs. The SA-β-gal assay was calculated using the following formula:
3
Colony Forming Unit Assay for Stem Cells
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The colony forming unit (CFU) assay was determined by re-plating 100 cells in a 35-mm dish (BD Bioscience), followed by 14 days of culturing at 37°C with 5% CO2 . Then, the cells were rinsed from growth media twice using DPBS -Ca2+, -Mg2+ (Invitrogen) and fixed with 100% methanol (Mallindkrodt, Hazelwood, USA, http://pharmaceuticals.covidien.com ) for 20 minutes at room temperature, followed by 3% crystal violet (Sigma Aldrich) staining. Next, the blue stain was rinsed out using tap water for four times until the dishes became colourless. The dishes were then inverted downwards on a clean cloth, and allowed to air-dry for several minutes. Stained colonies with sizes larger than 2 mm were counted. The CFU of ACSs was then calculated using the formula;
4
Senescence Sorting by CellTrace CFSE
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After senescence induction, the cells were washed with DPBS+Ca2+/Mg2+(Gibco) and incubated with 5 µM CellTrace CFSE dye (Thermo Fisher) diluted in DPBS+Ca2+/Mg2+ for 20 min in the incubator. Afterwards, the staining solution was removed, and the cells were washed with culture medium and cultured in fresh medium for 10 (senescent cells) or 4 (control cells) days in the incubator. After CellTrace CFSE staining, the cells were collected, centrifuged, resuspended in pre-sort buffer (BD Biosciences, Franklin Lakes, NJ, USA, cat. number: 563503) at a concentration of 1 × 106 cells/mL and sorted using the Sony SH800S Cell Sorter. The gates were adjusted to separate senescent and senescence-escaped cells by using control and senescent cells stained with CellTrace CFSE.
5
Senescence Induction and Cell Sorting
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After senescence induction, the cells were washed with DPBS+Ca 2+ /Mg 2+ (Gibco) and incubated with 5 µM CellTrace CFSE dye (Thermo Fisher) diluted in DPBS+Ca 2+ /Mg 2+ for 20 min in the incubator. Afterwards, the staining solution was removed, and the cells were washed with culture medium and cultured in fresh medium for 10 (senescent cells) or 4 (control cells) days in the incubator. After CellTrace CFSE staining, the cells were collected, centrifuged, resuspended in pre-sort buffer (BD Biosciences, Franklin Lakes, NJ, USA, cat. number: 563503) at a concentration of 1 × 10 6 cells/mL and sorted using the Sony SH800S Cell Sorter. The gates were adjusted to separate senescent and senescence-escaped cells by using control and senescent cells stained with CellTrace CFSE.
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