Two groups of cells were treated with basic DMEM and ox-LDL (50 mg/L) for 24 h. HCASMCs (1 × 106) were detached from six-well plates using 0.25% trypsin–ethylene diamine tetraacetic acid (EDTA) (Beyotime, Shanghai, China) (Corning, NY, United States). Digested cells were collected and washed with phosphate buffered saline (PBS; Beyotime, Shanghai, China), centrifuged twice (2,000 × g rpm, 5 min), fixed in 70% ethanol, and kept at 4°C overnight. Next, the cells were centrifuged at 1,000 × g rpm for 3 min and washed with PBS to remove residual ethanol. The centrifugation pellet was then resuspended in 500 μl of working fluid containing 10% RNase A and 90% propidium iodide (KeyGen BioTECH, Nanjing, China) for 60 min in the dark. The red fluorescence of propidium iodide was analyzed at 488 nm. Cell cycle analysis was conducted with a flow cytometer (Gallios Flow Cytometer; Beckman Coulter, United States), and data were processed with the corresponding software (Kaluza for Gallios; Beckman Coulter, United States).
Kaluza for gallios
Manufactured by Beckman Coulter
Sourced in United States
Kaluza for Gallios is a data analysis software designed to work with the Gallios flow cytometer. It provides tools for data acquisition, analysis, and visualization.
Automatically generated - may contain errors
Lab products found in correlation
Gallios flow cytometer, by Beckman Coulter (4 mentions)
Rnase a, by Keygen Biotech (2 mentions)
Propidium iodide, by Keygen Biotech (2 mentions)
Trypsin, by Beyotime (1 mentions)
Cd105, by BioLegend (1 mentions)
Six well plate, by Corning (1 mentions)
Phosphate buffered saline (pbs), by Beyotime (1 mentions)
Trypsin edta, by Beyotime (1 mentions)
4 protocols using kaluza for gallios
1
Cell Cycle Analysis of Ox-LDL Treated HCASMCs
2
Characterization of Mesenchymal Stem Cells
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Cells were cultured in DMEM with 10% FBS, and MSC characterization was performed in passage 6. Reverse transcription polymerase chain reaction (RT-PCR) was used to identify stem cell markers. To confirm that the criteria of the International Standard of MSC were met, we examined the expression of specific markers in cultured cells using fluorescence activated cell sorting (FACS) [25] (link). The cultured cells were stained according to the protocol provided by the manufacturer (Beckman Coulter, USA). Briefly, the cultured cells were detached and washed several times with PBS. The detached cells were divided into groups (approximately 1 × 106 cells) and stained with specific antibodies. The antibodies used for FACS analysis were for cell surface markers, including CD105, CD90, CD73, CD45, CD34 and CD14 (BioLegend, CA, USA, anti-human). The antibodies were conjugated to the fluorescein dye, with IgG1 PE or Fluorescein isothiocyanate (FITC) used as control dyes. The analysis was conducted with a FACS cytometer (Gallios Flow Cytometer; Beckman Coulter, USA) and software (Kaluza for Gallios; Beckman Coulter, USA). Specific media and RT-PCR were used to identify osteogenesis, adipogenesis, and chondrogenesis.
3
Immunophenotyping of Fetal Wharton's Jelly-Derived Mesenchymal Stem Cells
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To determine the immunophenotype of fWJ-MSCs, cells were stained with specific antibodies for fluorescence-activated cell sorter (FACS) analysis, following the protocol provided by the supplier (Beckman Coulter, Brea, CA, USA). Briefly, the fWJ-MSCs were trypsinized and washed several times with PBS at passage 5. The suspended cells were aliquoted (approximately 1 × 106 cells) for specific antibody staining. The cells were immunostained with the antibodies shown in the Table 2 . The antibodies were conjugated with fluorescein isothiocyanate (FITC) or phycoerythrin (PE). Analysis was determined by the use of FACS (Gallios Flow Cytometer; Beckman Coulter, Brea, CA, USA) and software (Kaluza for Gallios; Beckman Coulter, Brea, CA, USA).
4
Cell Cycle Analysis of HCASMCs
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Three groups of cells were cultured with DMEM basic medium, ox-LDL (50 mg/L), and SBP (2.5 mg/L), respectively, for 24 h. HCASMCs (1 × 106) were collected using 0.25% trypsin–EDTA (Beyotime, Shanghai, China) from the six-well plates (Corning, NY, USA). Detached cells were clustered and washed with PBS (Beyotime, Shanghai, China) and then centrifuged twice (2,000 rpm, 5 min). The HCASMCs were fixed in 70% ethanol overnight at 4°C. The fixed HCASMCs were centrifuged for 3 min at 1,000 rpm and resuspended with PBS for removal of residual ethanol. The centrifuged cells were then incubated with 500 μl of working fluid including 10% RNase A and 90% propidium iodide (KeyGen BioTECH, Nanjing, China) for 1 h in the dark. The cell cycle analysis was processed with a flow cytometer (Gallios Flow Cytometer; Beckman Coulter, USA), and data were analyzed with the suggested software (Kaluza for Gallios; Beckman Coulter, USA).
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