A FITC–annexin V apoptosis detection kit (BD Pharmingen, Franklin Lakes, NJ, USA) was used to measure apoptosis by flow cytometry, according to the manufacturer's protocol. HUVECs were cultured and treated as described in “HUVEC culture”. Briefly, 1 × 106 cells were collected, washed with ice-cold PBS, and double-stained with 5 μL of FITC–nnexin V and 5 μL of propidium iodide for 20 min at 25 °C in the dark. The fluorescence intensity was quantified using a flow cytometer (FlowSight, Merck Millipore) equipped with a 488 nm argon laser using a band pass filter of 530 nm. Data were acquired, analyzed, and plotted using the IDEAS software (FlowSight, Merck Millipore).
Flowsight
Manufactured by Merck Group
Sourced in United States, Germany
FlowSight is a flow cytometry instrument manufactured by the Merck Group. It is designed to analyze and sort cells and particles in a fluid stream. The FlowSight utilizes laser technology and digital signal processing to detect and measure various properties of the samples, including size, granularity, and fluorescence. It provides accurate and reliable data for research and diagnostic applications.
Automatically generated - may contain errors
Lab products found in correlation
Fitc annexin 5 apoptosis detection kit, by BD (1 mentions)
Dcfh da probe, by Merck Group (1 mentions)
Annexin 5 fitc pi apoptosis detection kit, by Keygen Biotech (1 mentions)
Live dead baclight, by Thermo Fisher Scientific (1 mentions)
Daf fm, by Thermo Fisher Scientific (1 mentions)
Facscalibur, by BD (1 mentions)
Pi solution, by Yeasen (1 mentions)
Annexin 5 fitc, by Merck Group (1 mentions)
Annexin 5 binding buffer, by Merck Group (1 mentions)
Inspire software, by Merck Group (1 mentions)
Mitosox red, by Thermo Fisher Scientific (1 mentions)
Mitosox red, by Merck Group (1 mentions)
Apoptosis assay kit, by BD (1 mentions)
Annexin 5 fitc kit, by Thermo Fisher Scientific (1 mentions)
Anti p65, by Abcam (1 mentions)
Human pluripotent stem cell transcription factor analysis kit, by BD (1 mentions)
Human msc analysis kit, by BD (1 mentions)
22 protocols using flowsight
1
Apoptosis Quantification in HUVECs
2
Intracellular ROS Evaluation in HUVECs
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The effects of folic acid on the intracellular ROS levels in HUVECs were detected by flow cytometry using a DCFH-DA probe (Sigma-Aldrich, St. Louis, MO, USA). HUVECs were cultured and treated as described in “HUVEC culture”. Cells were incubated with 10 μM DCFH-DA for 30 min in the dark, in a humidified 5% CO2 atmosphere. The cells were washed thrice with PBS to remove the extracellular DCFH-DA fluorescence probes, collected by centrifugation, and then suspended in PBS. The fluorescence intensity was quantified by a flow cytometer (FlowSight, Merck Millipore) equipped with a 488-nm argon laser using a band pass filter of 530 nm. The mean fluorescence intensity was acquired, analyzed, and plotted using the IDEAS software (FlowSight, Merck Millipore). Data are reported as DCF fluorescence, which indicates the percentage of control cells.
3
Apoptosis Evaluation by Flow Cytometry
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Cells were collected and processed according to the instructions for the Annexin‐V FITC/PI Apoptosis Detection Kit (KGA107, KeyGEN BioTECH). A flow cytometer was used to analyze the cells (Flow Sight, Millipore).
4
Optimizing A. chroococcum Cultivation
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Every 2 h, 1 mL of bacterial suspension was aseptically removed from a shake flask culture condition, the final optimal fermentation conditions and serially diluted 10-fold in PBS. One mL of the diluted solution and 3 mL LIVE/DEAD Baclight TM (L7012, Thermo) staining reagent were mixed and then incubated for 30 min in the dark. The total number of viable A. chroococcum was then measured by multispectral imaging flow cytometry (FlowSight, Millipore, USA). The culture duration and the total number of viable A. chroococcum were considered as the abscissa and ordinate, respectively, and the growth curve was generated (Calvert Meredith et al., 2008; Dalton and Postgate, 1969) .
Single-factor test. In the single factor test, a fixed culture time of 48 h was used, and the remaining factors (liquid solubility, initial pH, inoculum size, liquid volume, temperature, and shaking speed) and levels were selected (Kang et al., 2014; Ren et al., 2014; Zhang et al., 2013) . Two parallel experiments were carried out at each level, and were repeated in triplicate. The total number of living A. chroococcum in the fermentation broth was analyzed using multispectral imaging flow cytometry.
Single-factor test. In the single factor test, a fixed culture time of 48 h was used, and the remaining factors (liquid solubility, initial pH, inoculum size, liquid volume, temperature, and shaking speed) and levels were selected (Kang et al., 2014; Ren et al., 2014; Zhang et al., 2013) . Two parallel experiments were carried out at each level, and were repeated in triplicate. The total number of living A. chroococcum in the fermentation broth was analyzed using multispectral imaging flow cytometry.
5
Fluorescent Labeling Analysis
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6
Multicolor Flow Cytometry for Cell Analysis
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Cells were acquired using a FlowSight® imaging flow cytometer (Amnis®, part of EMD Millipore, MA, USA). Cell debris and dead cells were identified using the aspect ratio and area of the cells and removed. Approximately 7000 cells were obtained during each analysis. Channel 7 was used to detect BV421, Channel 3 to acquire PE, and Channel 11 to detect APC. Single colour control samples were compensated using a compensation matrix (.rif) and converted to data analysis files (.daf) and compensated image files (.cif) using the same settings. The data were analysed using Ideas software, version 6.2.
7
PD-L1 Expression Analysis by Flow
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PD-L1 staining used standard protocols with human monoclonal antibody: PE-anti-PD-L1 (BD Biosciences). Data were acquired by a flow cytometer (FACSCalibur, BD Biosciences) and flow imaging analysis (Flowsight, Merck Millipore).
8
Cell Cycle Analysis of Rapha Myr Extract
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Cell cycle analysis was performed on untreated and 24 h treated cells with Rapha Myr® extract (0.5–1.25–2.5% v/v), according to Malfa et al., 2019 [70 (link)]. In order to assess cell cycle state, 1 × 106 cells were stained with propidium iodide (PI, 50 ng/mL) and analyzed by the FlowSight® (Amnis®, part of EMD Millipore) imaging flow cytometer. The cell distribution in each cell-cycle phase was determined by using IDEAS 6.0 image analysis software (Amnis Inc., Seattle, WA, USA).
9
Isolation and Analysis of Murine Lymphocytes
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Mesenteric lymph nodes were isolated from each group of mice, and lymphocytes were isolated by a mouse lymphocyte isolation kit. Lymphocyte suspensions were prepared and adjusted to 2.0 × 106 cells per vial in RPMI 1640 medium containing 10% fetal bovine serum (FBS). Then prepared cells were labelled for 20 min in ice bath using the fluorescein isothiocyanate (FITC)-conjugated anti-mouse CD4 antibody (ab), phycoerythrin (PE)-conjugated anti-mouse CD25 ab, and allophycocyanin (APC)-conjugated anti-mouse Fork head box (Fox) P3 ab. The labeled samples were analyzed by flow cytometry (FlowSight, Merck Millipore), and data was analyzed using FlowJo software (Tree Star Inc. Ashland, OR, United States).
10
Heterocyst Identification via Flow Cytometry
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A minimum number of 10,000 cells were sampled on an image-based flow cytometer (Merck Amnis FlowSight) and analyzed using analysis software IDEAS. Single cells were identified based on the first gating with area scatter and aspect ratio scatter parameters. Thereafter, the cells were sub-gated with intensity scatter on YFP fluorescence channel, at 488nm channel (for YFP, FITC, AF488, and GFP) isolating all cells displaying fluorescence in the said channel. In order to isolate heterocysts, in case any other cells were displaying fluorescence in the YFP channel, a third step gating was performed. Based on the 488nm channel data, the cells were sub-gated with the intensity scatter on 745-800nm channel, indicating autofluorescence. The targeted single heterocyst cells were selected based on significantly decreased autofluorescence compared to vegetative cells.
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