PMN apoptosis was assessed: (1) in EDTA whole blood directly after being drawn at the defined time points post polytrauma or directly after being drawn from male healthy volunteers, which then was stained with FITC-labeled Annexin V and 7-AAD, lysed by FACS lysing solution (BD Pharmingen, San Diego, CA), washed twice, and fixed (1% paraformaldehyde plus 0.1% sodium azide) (as previously described [21 (link)]) followed by flow cytometric analyses; and (2) in isolated PMN (from citrated blood, see above) for the indicated in vitro studies. PMNs were stained for Annexin V/7-amino-actinomycin D (7-AAD) following the manufacturer’s instructions. Briefly, cells were adjusted to 2 × 106 cells/mL in binding buffer and stained with FITC-labeled Annexin V and 7-AAD on ice for 20 min. Cell gating included separation of the granulocytes and monocytes by the typical forward and side light scatter profiles and additional staining with anti-CD45/anti-CD14 (Leukogate; Beckman Coulter, Kreefeld, Germany). Fluorescence measurement was performed in both cases on a FACSCanto™ II flow cytometer using FACSDiva software v9.0 (BD Biosciences, Franklin Lakes, NJ, USA). Cells were classified as viable (Annexin Vlow/7-AADlow), early apoptotic (Annexin Vhigh/7-AADlow), and late apoptotic or necrotic (Annexin Vhigh/7-AADhigh).
Facsdiva software v9
Manufactured by BD
Sourced in United States
FACSDiva software v9.0 is a flow cytometry analysis software designed for use with BD instruments. It provides tools for data acquisition, analysis, and visualization. The software supports a range of features for managing and processing flow cytometry data.
Automatically generated - may contain errors
Lab products found in correlation
Facscanto 2 flow cytometer, by BD (3 mentions)
Facs lysing solution, by BD (1 mentions)
Pi rnase staining buffer, by BD (1 mentions)
Accuri c6 flow cytometer, by BD (1 mentions)
Lsr 2 flow cytometer, by BD (1 mentions)
Expifectamine enhancer, by Thermo Fisher Scientific (1 mentions)
Apc annexin 5 apoptosis detection kit with pi, by BioLegend (1 mentions)
Facscalibur, by BD (1 mentions)
Annexin 5 fitc pi apoptosis detection kit, by BD (1 mentions)
Lsrfortessa flow cytometer, by BD (1 mentions)
Annexin 5 pi, by BD (1 mentions)
Lsrii cytometer, by BD (1 mentions)
Apc brdu flow kit, by BD (1 mentions)
Facscanto 2 cytometer, by BD (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
10 protocols using facsdiva software v9
1
PMN Apoptosis Assessment in Polytrauma
2
Flow Cytometric Analysis of Rhodamine 123 Uptake
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Cells were treated with Etn for different time intervals. Subsequently, cells were incubated with rhodamine 123 (Sigma-Aldrich, #R8004) for 1 h at 37 °C. Cells were washed with PBS and analyzed by flow cytometry (excitation wavelength: 488 nm; emission wavelength: 515-575 nm). After the acquisition of 10000 events, the side scatter (SSC) was plotted against the forward scatter (FSC). Appropriate gating was performed to select positive cells while excluding debris and doublets. Flow cytometry data were analyzed using FACSDiva software v9.0 (BD Biosciences).
3
Fas/FasL Expression After Polytrauma
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EDTA blood from healthy volunteers and polytraumatized patients drawn at the indicated timepoints after trauma was used. Cells in EDTA whole blood were stained with a PE-labeled anti-FasL and PE-labeled anti-Fas antibody (Bio-Rad, Hercules, CA, USA) for 20 min. After lysis of red blood cells with FACS Lysing Solution (Cat. Nr. 349292, BD Biosciences, Franklin Lakes, NJ, USA), cells were resuspended in DPBS, and fluorescence measurement was performed on a FACSCantoTM II flow cytometer using FACSDiva software v9.0 (BD Biosciences, Franklin Lakes, NJ, USA).
4
Cell Cycle Analysis of Exosome-Treated MAC-T Cells
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MAC-T cells were treated with PBS or blood-derived exosomes isolated from the D30 and D250 periods (50 μg/mL). After 48 h, cells were collected, fixed in 75% ethanol, and stored overnight at −20 °C. They were then resuspended in 500 μL PI/RNase staining buffer (BD Biosciences, Franklin Lakes, NJ, USA) and incubated at 37 °C for 30 min. Flow cytometric cell cycle analysis was performed on the BD Accuri™ C6 flow cytometer and FACSDiVa software v9.0 (BD Biosciences).
5
SARS-CoV-1 S Protein mAb Screening
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Expi293F cells (Thermo Fisher, Cat# A14527) were transiently transfected with SARS-CoV-1 S-protein expression vectors (pcDNA3.3_CoV1_D28) using Expifectamine Enhancer according to the manufacturer’s protocol (Thermo Fisher). Two days later, to exclude dead cells from analysis, Expi293F were harvested, dispensed into a 96-well plate (3 × 105 cell/well), and stained for 30 min at room temperature (RT) with Live/Dead Fixable Aqua reagent (Invitrogen; Thermo Scientific) diluted 1:500. Following Live/Dead staining, cells were washed with PBS and incubated with nAbs candidates for 40 min at RT. Next, to identify the SARS-CoV-1 S protein mAbs binders, cells were washed and stained with the Alexa Fluor 488-labeled secondary antibody Goat anti-Human IgG (H + L) secondary antibody (Invitrogen) diluted 1:500. After 40 min of incubation, labeled cells were washed, resuspended in 150 μl of PBS, and analyzed using the BD LSR II flow cytometer (Becton Dickinson). Cells incubated with the SARS-CoV-1 nAb binder (S309) or incubated only with the secondary antibody were used as positive and negative controls respectively. Data were collected with the BD FACSDiva Software v9.0 (BD Biosciences) and analyzed with FlowJo™ Software (version 10).
6
Apoptosis Assay in ARPE-19 Cells
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The rate of apoptosis was determined in ARPE-19 cells using the APC Annexin V Apoptosis Detection Kit with PI (BioLegend, San Diego, CA, USA). The assay is based on fluorochrome-labeled annexin V and propidium iodide (PI) to distinguish between early apoptotic, late apoptotic, and necrotic cells. Non-transfected and BDNF-transfected ARPE-19 cells were seeded into 24-well cell culture plates at a density of 1.2 × 105 cells. After 96 h, cells were either treated with 150 µM H2O2 for 24 h or left untreated. Cells were stained as described in the manufacturer’s protocol and analyzed using the BD FACSCanto II flow cytometer along with BD FACSDiva software v9.0 (BD, Franklin Lakes, NJ, USA).
7
Apoptosis Analysis of Doxorubicin-Treated 4T1 Cells
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The 4T1 cells were cultured in 6-well plates (1×105 cells per well) for 12 h at 37°C, and when cell proliferation reached 60–70%, the original medium was sucked out and the cells continued to be cultured with fresh medium containing CNPs@DOX solution (5 mg/l DOX) and DOX solution (5 mg/l) for 24 h at 37°C. The cells were then trypsinized, washed with PBS and collected (1×106 cells). An annexin V-FITC/PI Apoptosis detection Kit (BD Biosciences) was used to stain apoptotic cells by mixing 100 µl cell suspension, 5 µl annexin V-FITC and 5 µl PI. The cells were incubated for 15 min at 37°C and 200 µl binding buffer (0.01 M HEPES, pH 7.4; 0.14 M NaCl; 2.50 mM CaCl2) was added to each suspension. Finally, apoptosis was measured using a flow cytometer (FACSCalibur™; BD Biosciences) and analyzed by BD FACSDiva™ Software v9.0.
8
Annexin V/PI Apoptosis Assay
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Cells were labeled with Annexin V/PI (BD Biosciences) based on the supplied protocol. Flow cytometry-based detection was performed for Annexin V and PI using a LSRFortessa Flow Cytometer (BD Biosciences; cat. no. 649225). Data were analyzed using BD FACSDiva Software v9.0 (BD Biosciences). Annexin V+/PI− cells were considered to be early apoptotic. Annexin V+/PI+ cells were considered to be late apoptotic. The percentages early and late apoptotic cells determined from 3 independent experiments are presented as the means ± SD.
9
Cell Cycle Analysis by BrdU Flow Cytometry
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The APC BrdU Flow Kit (Becton Dickinson, Franklin Lakes, NJ, USA) was used for flow cytometric analysis of the cell cycle according to the manufacturer’s protocol. After the respective treatment (either cytokines, drugs, or controls), BrdU solution was added for another 3 h into the medium of the cells (10 µL of 1 mM BrdU per mL culture medium). Then, both the adherent as well as the detached cells in the supernatant were collected and counted. A total of 5 × 105 cells/well were placed into a 96-deep-well plate, fixed, permeablized, and stained according to the manufacturer’s protocol. Flow cytometric measurements were performed on an LSRII cytometer in combination with the FACSDiva software V. 9.0, and the derived data were analyzed using the FlowJo software V. 10.8.0 (all from Becton Dickinson).
10
Propidium Iodide Viability Assay
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Cell viability was assessed after thawing (P0) or after dissociation (P6 or fresh samples) by adding 5 μL of propidium iodide (1 mg/mL, Sigma Aldrich) to 200 μL of cell suspension 1 min before the analysis. The analyses were performed on a FACSCanto II cytometer (Becton Dickinson, Le Pont de Claix, France) and acquired using the FACSDiva software V9.0 (Becton Dickinson, San Jose, CA, USA). The fluorescence was evaluated using a 488-nm excitation laser and a 650 ± 13-nm bandpass emission filter.
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