Flow cytometry (FACSCalibur, Becton Dickinson, CA, USA) was used to detect hFSSCs’ surface antigens (105 cells per marker). Briefly, hFSSCs at approximately 90% confluency were trypsinized and fixed in 3.7% formaldehyde solution. All antibodies were diluted (1:100) with 1% bovine serum albumin (BSA). Cells were labeled with FITC-conjugated anti-CD73, CD90, CD105, Oct4, and Sox2 (BioLegend, San Diego, CA, USA) at 4 °C for 1 h. Appropriate isotype-matched antibodies were used as negative controls (BD Biosciences, San Jose, CA, USA). Data from 10,000 viable cells were acquired. List mode files were analyzed by FCS Express software (BD Biosciences).
Fcs express software
Manufactured by BD
Sourced in United States
FCS Express is a software for the analysis and visualization of flow cytometry data. It provides tools for data import, gating, statistics, and graphical representation of flow cytometry experiments.
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Lab products found in correlation
Facscalibur, by BD (1 mentions)
Pi apoptosis detection kit, by BD (1 mentions)
Facscan, by BD (1 mentions)
Pe conjugated anti mouse ab, by BD (1 mentions)
Gelatin veronal buffer gvb, by Merck Group (1 mentions)
Accuri c6 plus, by BD (1 mentions)
Inverted fluorescence microscope, by Zeiss (1 mentions)
Donkey anti rabbit alexa 488, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions)
Annexin 5 apc, by BioLegend (1 mentions)
Facscanto 2 flow cytometer, by BD (1 mentions)
Facscanto flow cytometer, by BD (1 mentions)
Jc 1 staining kit, by Beyotime (1 mentions)
Dcfh da, by BD (1 mentions)
Dcfh da, by Beyotime (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Lsrii flow cytometer, by BD (1 mentions)
Facsdiva, by BD (1 mentions)
Trypsin solution, by GE Healthcare (1 mentions)
Fitc annexin 5 apoptosis detection kit, by BD (1 mentions)
12 protocols using fcs express software
1
Flow Cytometric Analysis of hFSSCs
2
Apoptosis Detection in Treated HUVECs
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Apoptosis in treated HUVECs was detected using an Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis detection kits (BD Biosciences; cat. no. BD 556547), following the manufacturer’s protocol. In brief, HUVECs were treated as aforementioned, and the culture medium and the cells were collected. Following two PBS washes, the cells were resuspended in 1X binding buffer (100 μl) at a density of 1×105 cells/ml, and then double stained with 5 μl Annexin V-FITC and 5 μl PI for 15 min in the dark. The percentage of apoptotic cells was determined using a FACScan flow cytometer and analyzed using FCS Express software (version 1.2; BD Biosciences). Each experiment was performed in triplicate.
3
Bacterial Complement Deposition Assay
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Gelatin veronal buffer (GVB, Sigma-Aldrich, St. Louis, USA) supplemented with 13.3% ΔC1f2 serum and 16.6% CEMf2 was preincubated with or without inhibitors on ice for 15 min. 75µL of prechilled mixture was added to 25µL of GVB containing 5 × 105 CFUs of bacteria in microplate wells. Plates were incubated for 30 min at 37°C in 5% CO2 with shaking and then placed on ice to stop complement deposition. After bacteria were washed with ice-cold GVB, surface bound C3 or C4b/C4c bound were stained using murine anti-human C3 mAb (ThermoFisher Scientific, Waltham, USA; LF-MA0132, clone 28A1) and PE-conjugated anti-mouse Ab (BD Biosciences, Franklin Lakes, USA), or FITC-conjugated murine anti-human C4 mAb (ThermoFisher Scientific, Walktham, USA), and detected by flow cytometry using BD Accuri C6 Plus (BD Biosciences, Franklin Lakes, USA) and FCS Express software (Pasadena, USA).
4
Characterization of Stem Cell Markers
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Flow cytometry and immunofluorescence were used to identify the characteristics of the cells and detect stem cell-related cell surface markers. For flow cytometry, the cells (106 cells/100 μl) were collected and incubated with monoclonal phycoerythrin (PE)-conjugated antibodies for CD29 (cat. no. 303003), CD31 (cat. no. 303105), CD34 (cat. no. 343505), CD44 (cat. no. 338807), CD45 (cat. no. 368509), CD49d (cat. no. 304303), CD73 (cat. no. 344003), CD90 (cat. no. 32810), CD105 (cat. no. 323205), HLA-DR (cat. no. 307605), SSEA-4 (cat. no. 330405), SOX-2 (cat. no. 656103) and OCT-4 (cat. no. 653703) (BioLegend, San Diego, CA, USA) for 30 min on ice. Appropriate isotype-matched antibodies were used as negative controls (BD Biosciences, San Jose, CA, USA). Data from 10,000 viable cells were acquired. List mode files were analyzed by FCS Express software (BD Biosciences). For immunofluorescence, cells growing on the glass slide were stained with anti-cytokeratin 19 (cat. no. ab52625, 1:200; Abcam, Cambridge, MA, USA) and secondary antibody (cat. no. A-11034, 1:200; Alexa 488, donkey anti-rabbit; Life Technologies, Carlsbad, CA, USA). Nuclei were stained with DAPI (Beyotime, Shanghai, China). Cells on the glass slide were photographed using an inverted fluorescence microscope (Carl Zeiss, Oberkochen, Germany).
5
Proliferation, Cell Cycle, and Death Assays
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Proliferation rate of MPM cells was analysed by MTT test: briefly, cells were plated in 96 wells plates at different concentrations, and assayed after 24, 48 and 72 hours. MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) was added and left on cells for 3 hours at 37°C and 5% CO2. The resulting intracellular purple formazan was solubilized with SDS and quantified by spectrophotometer at λ = 550/650 nm. Cell cycle analysis was performed on G1 synchronized REN cells. Cells were starved in DMEM without FBS for 12 hours, and then in PBS plus 0,5 mM MgCl2, 10 mM D-Glucose, 1 mM CaCl2 for 3 hours. At the indicated time points, cells were fixed, stained with propidium iodide (PI) and acquired on a BD FACS CANTO II flow cytometer. Cell cycle analysis was performed using the FCS Express software (BD). Cell death detection was performed using APC-Annexin V (BioLegend). Each experiment was done at least in triplicate.
6
Mitochondrial Membrane Potential Assay
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The MMP in HUVECs cells was detected using a 5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) staining kit (Beyotime Institute of Biotechnology; cat. no. C2006). The cells were harvested, washed twice with PBS and resuspended in 10 μM JC-1 solution at 37°C for 20 min. Next, the JC-1 staining solution was removed and the cells were washed and resuspended in PBS. The fluorescence values were then analyzed with a FACSCanto flow cytometer and FCS Express software (version 1.2, BD Biosciences). The loss of MMP was reflected by the decrease in red fluorescence from the JC-1 aggregates and the increase in green fluorescence from the JC-1 monomers.
7
Intracellular ROS Measurement by DCFH-DA
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The intracellular ROS generation was analyzed by 2′,7′-dichlorofluorescindiacetate (DCFH-DA; Beyotime Institute of Biotechnology), a ROS-sensitive fluorescent dye, followed by flow cytometry. Briefly, following relevant treatment or transfection, cells were treated with RIPA lysis buffer and washed with cold PBS twice. Next, cells were stained with 10 μM 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) in the dark for 20 min at 37°C, and the fluorescence intensity was detected using a FACSCalibur flow cytometer (488 nm excitation/521 nm emission) and analyzed with FCS Express software (version 1.2; BD Biosciences).
8
Isolation and Characterization of Porcine MSCs
9
hAMSC Immunophenotypic Characterization
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The immunophenotype of hAMSCs cultured in either hAMSCs culture conditions or EC culture conditions was analyzed using flow cytometry. A total of 1×106 cells were detached from culture dishes using trypsin solution (Hyclone; GE Healthcare Life Sciences) and stained with 5 µl antibodies against cluster of differentiation (CD)31 (cat. no 303105), CD34 (cat. no 343505), CD73 (cat. no 344003), CD90 (cat. no 32810) and CD105 (cat. no 323205) (all undiluted; BioLegend, Inc., San Diego, CA, USA). Immunoglobulin G of the appropriate isotype was used as negative control. Data from 10,000 viable cells were acquired. List mode files were analyzed by FCS Express Software (version 3; BD Biosciences, Franklin Lakes, NJ, USA).
10
Cobalt-Induced Apoptosis Analysis
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Following 48 h incubation in serum-free DMEM with 300 µM cobalt (II) chloride, a FITC Annexin V Apoptosis Detection kit (BD Biosciences) was used to double stain cells with FITC-Annexin V and propidium iodide according to the manufacturer's protocols. A total of 2.5×105 cells were seeded into 6-well plates for 48 h and removed using trypsin without EDTA, stained cells were analyzed using a flow cytometer (BD Biosciences). FCS Express Software (version 3; BD Biosciences) was used to observe cell apoptosis. In the graphs, cells were distinguished as dead, living, early apoptotic and late apoptotic cells. The aggregate of early and late apoptotic cells was regarded as an observation index to compare the experimental and negative groups. Each experiment was performed at least three times.
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