Pyroptotic cell death was examined according to a previously published protocol (37 (link)). Briefly, the cultured BMMSCs were stained with caspase-1 antibody (D-3), FITC (sc-392736; Santa Cruz Biotechnology), and propidium iodide (PI) for 1 h and fixed with 4% paraformaldehyde on ice for 10 min. Following washing with Hanks’ balanced salt solution (Sigma-Aldrich), the cells were analyzed with a BD Accuri C6 Plus cell analyzer (BD Biosciences).
Accuri c6 plus cell analyzer
Manufactured by BD
Sourced in United States
The BD Accuri™ C6 Plus Cell Analyzer is a compact and easy-to-use flow cytometry system designed for various research applications. It provides automated sample acquisition, data analysis, and reporting capabilities.
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7 protocols using accuri c6 plus cell analyzer
1
Pyroptotic Cell Death Quantification
2
Characterizing Gingival Immune Cells
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Cells isolated from gingival tissues of all mice were counted, washed, and resuspend in 100 μL cold FACS buffer (PBS with 2% FBS and 1 mM EDTA). Appropriate antibodies combination (PerCP5.5-conjugated anti-CD45 and APC-conjugated anti-CD11b or PE-conjugated anti-CD4; PE-conjugated anti-IL-17α and APC-conjugated anti-CD11b) were added into the cell suspension for 30 min on ice. Cells were washed twice using cold FACS buffer. The IL-17α-positive and CD11b-positive cell population were analyzed using BD Accuri™ C6 Plus Cell Analyzer (BD Biosciences, San Jose, CA, USA).
3
Lentiviral Infection and Cellular Assays
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Cells were infected with a lentiviral vector carrying a shRNA targeting circZDHHC11 or a transgene (for overexpression of miR-150 or circZDHHC11) together with a reporter gene. We aimed at infection percentages of 10–50%. Cells infected with lentiviral vectors containing non-targeting or scrambled sequences were used as controls. Vectors contained GFP, RFP or BFP, and these reporters were used to follow the percentages of infected cells by flow cytometry over time on a BD Accuri C6 Plus Cell Analyzer (BD Biosciences), Calibur flow cytometer (BD Biosciences) or NovoCyte Quanteon flow cytometer (Agilent Technologies, Santa Clara, CA, USA). Percentages of infected cells were measured at day 4 post-transfection and monitored tri-weekly for three weeks. Data were analyzed using the FlowJo software (version 10, Treestar, BD Biosciences). To determine the effect on cell growth, the percentage of GFP/RFP/BFP positive cells at day 4 was set to 100% and the fold difference relative to this starting point was calculated for each time point. The mixed model analysis was performed as previously described to compare the difference of relative GFP/RFP/BFP percentages at day 22 post-transfection1 (link).
4
Quantifying Transient Transfection Efficiency
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The percentages of GFP positive cells were measured on the BD Accuri™ C6 Plus Cell Analyzer (BD Biosciences, Franklin Lakes, NJ, USA) at day 4 post-transfection and monitored tri-weekly for three weeks. Data were analyzed using FlowJo software (version 10, Treestar, Ashland, OR, USA). To determine the effect on cell growth, the percentage of GFP positive cells at day 4, 6 or 8 (depending on the time reaching the maximal GFP percentage which varied per construct and cell line) was set to 100% and the fold difference relative to this starting point was calculated for each time point. To determine significant differences in the GFP assays, we used mixed model analysis as described previously [11 (link)].
5
Apigenin Modulates Esophageal Cell Apoptosis
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Human esophagus cells were seeded into 6-well plates (3 × 105 cells/well) and treated with apigenin (0 µM vehicle control; 0.3, 1, 3, and 10 µM), with or without IL-6 (50 ng/µl). After 48 h, cells were harvested and stained with annexin V–fluorescein isothiocyanate (FITC) and propidium iodide (PI) for 15 min in the dark. BD ACCURI C6 PLUS Cell Analyzer was used to measure fluorescence intensity in FITC (FL1, 533 nm) and PI (FL2, 585 nm) channels. The early apoptotic cells (annexin V-positive only) and late apoptotic cells (annexin V- and PI-positive) were quantified and analyzed with the FlowJo 10.0.7 software.
6
Quantifying Apoptosis in Tumor Cells
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Flow cytometry was used to analyze the apoptotic rate of tumor cells that had received different treatments. Annexin V Apoptosis Detection Kit II (556570; BD Biosciences) was used for incubation with the collected cells to detect the apoptotic rate. The results were detected and obtained by BD AccuriTM C6 Plus cell analyzers (BD Biosciences). FlowJo 10.4 (FlowJo, LLC) was used to analyze the final results.
7
Quantification of Cell Death
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Cells were seeded into six-well plates and cultured in medium with or without glucose and appropriate drugs. Afterward, the cells were collected for analysis of dead (PI-positive) cells by FACS using BD AccuriTM C6 Plus cell analyzers (BD Biosciences, USA). The results were then analyzed using FlowJo 10.4 software.
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