Flow cytometry was used to detect the apoptosis of HL-60 cells affected by METTL14 and miR-1306-5p. FACSVerse flow cytometry (BD Biosciences) was used for examination. Briefly, Annexin V-fluorescein isothiocyanate (FITC) Apoptosis Detection kit (HS-SJ069, Hasenbio) was used to detect the apoptosis at 48 h after transfection. Transfected HL-60 cells resuspended in binding buffer (500 μl) were stained with PI (5 μl, 50 μg/ml) and Annexin V-FITC (10 μl). The stain process was in the dark and lasted for 15 min at room temperature. FACSVerse flow cytometry (BD Biosciences) was used for examination.
Facsverse flow cytometry
Manufactured by BD
Sourced in United States
The FACSVerse flow cytometry system is a compact and versatile instrument designed for a wide range of flow cytometry applications. It utilizes state-of-the-art optics and electronics to provide high-performance data acquisition and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Fcr blocking reagent, by Miltenyi Biotec (3 mentions)
Propidium iodide, by BD (3 mentions)
Facsuite software, by BD (3 mentions)
Annexin 5 fitc apoptosis detection kit, by Immunostep (2 mentions)
Annexin 5, by BD (2 mentions)
Annexin 5, by BioLegend (2 mentions)
Fluo 4 am, by Thermo Fisher Scientific (2 mentions)
Rnase a, by Beyotime (1 mentions)
Cytometric bead array cba mouse inflammation kit, by BD (1 mentions)
Mouse anti human cd45 antibody, by BD (1 mentions)
Mitosox red, by Thermo Fisher Scientific (1 mentions)
Mitotracker red cmxros, by Thermo Fisher Scientific (1 mentions)
Anti cd38 pe cy7, by BD (1 mentions)
Anti cd161 apc, by BD (1 mentions)
Anti cd3 apc cy7 sp34 2, by BD (1 mentions)
Anti cd8 percp cy5, by BD (1 mentions)
Anti cd4 fitc, by BD (1 mentions)
Dhr123, by BD (1 mentions)
Dhr123, by Merck Group (1 mentions)
Tmre mitochondrial membrane potential assay kit, by Abcam (1 mentions)
51 protocols using facsverse flow cytometry
1
Apoptosis Analysis of HL-60 Cells
2
Flow Cytometry Cell Staining Protocol
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All samples were analyzed by FACSVerse™ flow cytometry (BD Bioscience, USA). Two blood samples (100 μl each) were stained according to the manufacturer’s instructions. Then, red-cell lysis buffer (1 ml) was added to each sample and incubated for 10 min followed by washing with Sorvall cell washer (Thermo Fisher Scientific, USA). Cells were blocked with FcR blocking reagent (Miltenyi Biotec, USA) for 30 min at 4°C to reduce nonspecific binding of antibodies to human FcR and then washed with PBS. The cells were then stained with antibodies using the fixable dead cell stain kit (Invitrogen, USA) for 30 min at 4°C and were then resuspended in 350 μl PBS and analyzed in a flow cytometry (FACSVerse™ flow cytometry, BD Bioscience, USA). Calibration and quality control for the instrument were carried out daily with the use of eight-color setup beads (BD Bioscience). All specimens were analyzed in duplicates with a coefficient of variation (CV) <5% by two independent technicians under the inter-laboratory quality control. The experiments were repeated if the results exhibited a CV >5% according to the instructions of BD Bioscience. The data were analyzed by FlowJo software (version 10, Tree Star, USA).
3
Quantifying SABG and Apoptosis in ADSCs
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To determine cellular levels of SABG, we used the FluoReporter lacZ Flow Cytometry Kit (Invitrogen) following the manufacturer’s instructions. In brief, we harvested cells from the cell culture plate and diluted them to 1000 cells/μl in a staining medium and introduced a 1:1 volume of fluorescein di-β-d -galactopyranoside (FDG) 2 mM working solution; cells were stained for exactly 1 min at 37°C. FDG loading was interrupted by adding 1.8 ml of ice-cold staining medium containing 1.5 μM propidium iodide. Fluorescence values were read by FACSVerse flow cytometry (BD Biosciences, San Diego, CA, USA) until 10,000 events were recorded.
Apoptosis was assessed using an Annexin V-FITC Apoptosis detection kit (Immunostep, Salamanca, Spain), following the manufacturer’s instructions. ADSCs were harvested and diluted to 1000 cells/μl in annexin-binding buffer. Then, 100 μl of aliquots of resuspended cells were placed into an appropriate flow cytometer tube and stained with 5 μl of annexin V–FITC and 5 μl of propidium iodide for exactly 15 min at 37°C in darkness. After incubation, 400 μl of 1× annexin-binding buffer was added. The values were read by FACSVerse flow cytometry (BD Biosciences, San Diego, CA, USA) until 10,000 events were recorded.
Apoptosis was assessed using an Annexin V-FITC Apoptosis detection kit (Immunostep, Salamanca, Spain), following the manufacturer’s instructions. ADSCs were harvested and diluted to 1000 cells/μl in annexin-binding buffer. Then, 100 μl of aliquots of resuspended cells were placed into an appropriate flow cytometer tube and stained with 5 μl of annexin V–FITC and 5 μl of propidium iodide for exactly 15 min at 37°C in darkness. After incubation, 400 μl of 1× annexin-binding buffer was added. The values were read by FACSVerse flow cytometry (BD Biosciences, San Diego, CA, USA) until 10,000 events were recorded.
4
Intracellular Calcium and Mitochondrial Membrane Potential Assessment
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The fluorescent probe Fluo 3-AM was used for the assessment of intracellular levels of Ca2+ and the lipophilic cationic dye 3,3′-dihexyloxacarbocyanine iodide [DiOC6(3)] was used for measuring disruption of the MMP (Δψm). Briefly, cells were exposed to a 5-Gy radiation dose and incubated for 72 h, then stained with 1 µM DiOC6(3) at 37°C for 15 min in the dark, and analyzed using FACSverse flow cytometry (BD Biosciences). For apoptosis analysis, cells were harvested and centrifuged at 800 rpm for 3 min following radiation treatment (48 h). Cells were carefully resuspended, and hen Annexin-V (5 µl) and propidium iodide (PI) (5 µl) (BD Biosciences) were added, and the cells were incubated at 37°C for 15 min in the dark. The stained cells were analyzed using FACSverse flow cytometry (BD Biosciences) and the Flowjo software v7.6.1 (FlowJo LLC). At least 10,000 cells per sample were analyzed, and duplicate analyses were performed at each time point.
5
Cell Cycle Analysis by Flow Cytometry
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The cells were cultured to a density of 90%, collected, washed 3 times with PBS, and fixed in 1 ml of 70% low-temperature ethanol at 4°C overnight. After washing with PBS, cells were stained with 0.5 ml propidium iodide (25 μl propidium iodide, 10 μl RNaseA, Beyotime, China). Following that, the cells were re-suspended and bathed in water at 37°C for 30 min. FACSVerse flow cytometry (BD, USA) was used to assess the cell cycle.
6
Cytokine Profiling in Macrophage Response
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To evaluate the production of different mediators in response to LPG and LPS, macrophage culture supernatants were collected after 48 h of incubation. Tumor necrosis factor alpha (TNF‐α), interleukin 6 (IL‐6), IL‐10, IL‐12p70, and MCP‐1 concentrations were determined using BD cytometric bead array (CBA) Mouse Inflammation Kit (BD Biosciences) according to the manufacturer's specifications. Flow cytometry measurements were performed on FACSVerse flow cytometry (BD Biosciences). The Cell‐Quest software package provided by the manufacturer was used for data acquisition and the FlowJo v. 7.6.4 (Tree Star Inc.) was used for data analysis. A total of 2500 events were acquired for each analysis. The results are representative of three experiments in duplicate. Nitrite (NO) concentrations were determined by the Griess reaction (Drapier et al., 1988 (link)).
7
Isolation and Identification of ILC2s
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ILC2s were defined as Lin−CD45+CD127+CD294+ cells. PBMCs (Peripheral Blood Mononuclear Cells) were isolated as described as follows. Heparinized peripheral blood was diluted with 1 : 1 phosphate-buffered saline (PBS) and layered over an equal volume of ficoll as per the manufacturer's instructions. After centrifuged, mononuclear cells were collected from the interface between the ficoll and plasma. The collected PBMCs were washed three times with PBS and resuspended in 200 μL PBS before further manipulation. Obtained PBMCs were enumerated. PBS was added to adjust the concentration of the suspension to 5 × 103/μL. 200 μL aliquot of the obtained PBMCs was incubated with mouse anti-human CD127 antibody (5 μL), mouse anti-human CD45 antibody (5 μL), mouse anti-human CD294 antibody (5 μL), and human lineage compound antibody (CD2, CD3, CD4, CD7, CD8, CD10, CD11b, CD14, CD19, CD20, CD56, and CD235a (20 μL)) (BD Biosciences, America) at room temperature in the dark for 40 minutes and analyzed by FACSVerse flow cytometry (BD Biosciences, America).
8
Mitochondrial Function and ROS Measurement in ATG7 Knockdown Cells
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For measurements of mitochondrial function ATG7 knockdown, K562 cells were stained with TMRM (a cell-permeant, cationic, red-orange fluorescent dye, Life Technologies, T-668) and MitoTracker® Red CMXRos (a reduced probe that does not fluoresce until it enters live cells, where it is oxidized and sequestered in functional mitochondria and starts emitting red fluorescence, Invitrogen, M-7512). For ROS measurements ATG7 knockdown or HCQ-treated cells were washed with phosphate-buffered saline (PBS; Gibco, 14190–094, 18912–014) and incubated with 5 μM MitoSox™ Red (Invitrogen, M36008) in PBS at 37°C in the dark for 30 min. Cells were then centrifuged (400 g for 5 min) and resuspended in PBS. Fluorescence was detected by FACSVerse flow cytometry (BD Biosciences, 651155, San Jose, CA, USA). For assessment of differentiation K562 and CML CD34+ cells were stained with fluorochrome-conjugated mAbs directed against CD34 (BD Biosciences, 555824), TFRC (BD Biosciences, 555537) and GYPA (BD Biosciences, 561776). Data analyses were performed using FlowJo software (Tree Star).
9
Evaluating Apoptosis in AGS Cells
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The flow cytometry assay was used to evaluate the apoptosis of AGS cells. AGS cells (3×105 cells/well) were seeded into a 6-well plate and incubated for 24 h. After washing the cells thrice with PBS, the medium containing ZC, ZT, ZTC, ZTC@M, and ZTC@M + US (TPZ 12 μg/ml and Ce6 2 μg/ml) was added to the plates, respectively, and cultured for 4 h. The US was applied at 1.0 MHz, 1.5 W/cm2 for 3 min. Next, supernatants and cells were collected and then were mixed with 500 µl of binding buffer, to which 5 µl of Annexin V-FITC and 10 µl of PI were added and incubated for 5 min in the dark. Finally, cell apoptosis was detected using FACSVerse flow cytometry (BD, United States).
10
Multicolor Flow Cytometry Immunophenotyping
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The staining procedure was the same as described before. In short, after thawing, samples were treated with FcR Blocking Reagent (Miltenyi Biotech, Germany) and stained accordingly with human anti-bodies (mAbs). They were anti-CD8-PerCP-Cy5.5, anti-CD3-APC-Cy7 (SP34–2), anti-CD4-FITC, anti-Vα 7.2-PE, anti-CD161-APC, anti-CD38-PE-Cy7, anti-CD279-PE-Cy7 and all of them were from BD Biosciences (Heidelberg, Germany). Then samples were washed. Acquisition was carried out by six-color flow cytometry using FACSVerse™ flow cytometry (BD Biosciences) with FACSuite software (BD Biosciences). Analyses of the data were made by FlowJo software (Tree Star, Ashland, OR, USA).
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