CMs derived from human-induced pluripotent stem cells (iCell® Cardiomyocytes2, Fujifilm/Cellular Dynamics International) were seeded onto 24-well cell culture plates coated with 380 µl of ready-to-use Geltrex™ basement membrane extracellular matrix (Geltrex, ThermoFisher Scientific) at a density of 1 × 105 cells cm−2 following instructions by the manufacturer. Briefly, vials of 5 × 106 hiPSC-CMs were thawed and seeded using the manufacturer's cell plating medium, which was exchanged by the manufacturer's maintenance medium 4 h after seeding. Viable cells were counted with a hemocytometer and trypan blue (Biowest) as a counter stain. The cells were cultured in an incubator (37°C/5% CO2), the medium was exchanged every 2 days and the cells assayed on the 7th day of culture.
Trypan blue
Manufactured by Biowest
Sourced in Belgium, United States
Trypan blue is a staining dye used in cell culture applications. Its core function is to distinguish between viable and non-viable cells by selectively staining only the non-viable cells.
Automatically generated - may contain errors
Lab products found in correlation
Geltrex, by Thermo Fisher Scientific (1 mentions)
Icell cardiomyocytes2, by Fujifilm (1 mentions)
Countess 2 automated cell counter, by Thermo Fisher Scientific (1 mentions)
Axioskop 40 fl, by Zeiss (1 mentions)
Wst 1, by Takara Bio (1 mentions)
Optizen pop, by Mecasys (1 mentions)
Mitomycin, by Merck Group (1 mentions)
U bottomed 96 well plates, by Corning (1 mentions)
2 3 bis 2 methoxy 4 nitro 5 sulfophenyl 2h tetrazolium 5 carboxanilide xtt assay kit, by Sartorius (1 mentions)
Ficoll solution, by Merck Group (1 mentions)
Fetal calf serum (fcs), by Merck Group (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
4 protocols using trypan blue
1
Cardiomyocyte Derivation and Culture
2
Comparative Cell Culture Protocols
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Vero cells were cultured in 25 cm2 cell culture flasks using Medium 199 with Earl's salt, 10% FBS, and 1% penicillin-streptomycin for group A and Medium 199 with Earl's salt, 10% HPL, 0.25 IU/mL heparin, and 1% penicillin-streptomycin for group B and then incubated at 37℃ in a 5% CO2 atmosphere.
Hep-2 Cells were seeded in 25 cm2 cell culture flasks using MEM Earl's media, 10% FBS, and 1% penicillin and streptomycin for group A and MEM Earl's media, 10% HPL, and 1% penicillin and streptomycin for group B and incubated in the presence of 5% CO2 at 37℃.
Cells were detached using trypsin ethylenediaminetetraacetic acid (EDTA; #BE17-161E, Lonza, Belgium) on day 2 and 5, mixed (1:1) with trypan blue (#L0990-100, Biowest), and counted using Invitrogen Countess II Automated Cell Counter (Thermo Fisher Scientific, Waltham, MA, USA) [13 ].
Hep-2 Cells were seeded in 25 cm2 cell culture flasks using MEM Earl's media, 10% FBS, and 1% penicillin and streptomycin for group A and MEM Earl's media, 10% HPL, and 1% penicillin and streptomycin for group B and incubated in the presence of 5% CO2 at 37℃.
Cells were detached using trypsin ethylenediaminetetraacetic acid (EDTA; #BE17-161E, Lonza, Belgium) on day 2 and 5, mixed (1:1) with trypan blue (#L0990-100, Biowest), and counted using Invitrogen Countess II Automated Cell Counter (Thermo Fisher Scientific, Waltham, MA, USA) [13 ].
3
Evaluating Cell Metabolic Activity
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To measure the impact of the samples on the metabolic activity, the cells were incubated for 24 h under the above-mentioned conditions. Then the sample-containing medium was removed, the cells were washed twice with PBS and 1 mL growth medium, containing 20 µL/mL 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-1; Clontech, Mountain View, CA, USA), was added. Afterwards, the cells were incubated further for 4 h.
Finally, for quantitative analysis, 800 µL WST-1 containing growth medium was centrifuged (1 min, 8000× g) to precipitate possible cell debris before measuring the absorbance at λ = 440 nm and λ = 690 nm (as background) (Mecasys Optizen-Pop, Daejeon, South Korea). The absorbance of the growth control was normalized to 100%, all other values are expressed relative to this (relative WST-activity).
To detect necrosis, 0.05% (final concentration) trypan blue (Biowest, Kansas City, MO, USA) was added, incubated for 5 to 10 min, the cells grown on cover slides were washed with PBS and then observed under the microscope (Axioskop 40 FL, Zeiss, Oberkochen, Germany) [25 ].
Finally, for quantitative analysis, 800 µL WST-1 containing growth medium was centrifuged (1 min, 8000× g) to precipitate possible cell debris before measuring the absorbance at λ = 440 nm and λ = 690 nm (as background) (Mecasys Optizen-Pop, Daejeon, South Korea). The absorbance of the growth control was normalized to 100%, all other values are expressed relative to this (relative WST-activity).
To detect necrosis, 0.05% (final concentration) trypan blue (Biowest, Kansas City, MO, USA) was added, incubated for 5 to 10 min, the cells grown on cover slides were washed with PBS and then observed under the microscope (Axioskop 40 FL, Zeiss, Oberkochen, Germany) [25 ].
4
Immunomodulatory Effects of HUCMSCs
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The immunomodulation activity of the HUCMSCs was demonstrated with MLR. Human PBMCs from two different donors (male, 25 and 40 years old) who provided informed consent were isolated from heparinized blood by gradient centrifugation with Ficoll solution (Sigma-Aldrich) at 400 × g for 20 min at room temperature. Stimulator PBMCs were prepared by treatment with mitomycin (Sigma-Aldrich) at 25 μg/ml for 30 min at 37°C. Cell count and viability were assessed by trypan blue (Biowest) dye exclusion and then used directly in the MLR. The HUCMSCs were plated in triplicate at passage 2 into U-bottomed 96-well plates (Corning) at 10 5 cells/ ml in 100 μl of FCS-DMEM and allowed to adhere to the plate for 1 to 2 h. Human responder (10 5 PBMCs) and an equal number of stimulator PBMCs were added to the wells in 100 μl of RPMI-1640 (Invitrogen) in 10% inactivated FCS (Sigma-Aldrich). The cultures were incubated at 37°C in 5% CO 2 for 5 days, and the suspended responder PBMCs were then counted using the 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5carboxanilide (XTT) assay kit (Biological Industries). To test the role of HLA-G in MLR, 3 μg/well of HLA-Gneutralizing antibodies (87G Abs) or control antibody (20 μg/ml) (Exbio Antibodies, Praha, Czech Republic) was added on the first day of the MLR cultures.
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