Fold-induction of apoptosis was evaluated using the FITC Annexin V Apoptosis Detection Kit with 7-AAD (Biolegend, CA, USA) according to the manufacturer’s instructions, after no irradiation for control cells and after 5 Gy irradiation for irradiated cells using Gulmay MP1-CP225 X-ray unit as described above. After irradiation, cells were incubated for 5 h, 24 h, 48 h, and 72 h. Trypsinized cell suspension was combined with the collected cell media to include dead cells and centrifuged to remove the trypsin and cell media. Early apoptotic (Annexin V-positive and 7AAD-negative), late apoptotic and/or necrotic (Annexin V-positive and 7AAD-positive), necrotic (Annexin V-negative and 7AAD-positive) and viable (Annexin V-negative and 7AAD-negative) cells were analyzed by FACSCanto II flow cytometer (BD Biosciences, CA, USA) within 10 min (Additional file 4 : Figure S2). Fold-induction of apoptosis was determined as the sum of early and late apoptotic (Annexin V-positive) cells at a specific time point, normalized to the sum of early and late apoptotic cells in control non-irradiated cells at 5 h. Fold-change of cell viability was determined as percentage of viable non-irradiated or 5 Gy irradiated cells at specific time point, normalized to the percentage of control non-irradiated viable cells at 5 h. The experiment was repeated 4 times.
7 aad
Manufactured by BD
Sourced in United States, Germany, United Kingdom, Denmark, Belgium, France, Canada, Japan
7-AAD is a fluorescent dye used for cell staining and analysis in flow cytometry. It binds to DNA and can be used to detect cell viability and apoptosis.
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Lab products found in correlation
Annexin 5 pe, by BD (96 mentions)
Annexin 5, by BD (86 mentions)
Facscalibur, by BD (79 mentions)
Facscanto 2, by BD (77 mentions)
Annexin 5 apc, by BD (58 mentions)
Annexin 5 fitc, by BD (57 mentions)
Annexin 5 binding buffer, by BD (42 mentions)
Lsrfortessa, by BD (37 mentions)
Facsdiva software, by BD (35 mentions)
Facscanto 2 flow cytometer, by BD (31 mentions)
Facscanto, by BD (31 mentions)
Facsaria, by BD (29 mentions)
Facsaria 2, by BD (28 mentions)
Facscalibur flow cytometer, by BD (25 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (25 mentions)
Binding buffer, by BD (24 mentions)
Accuri c6, by BD (22 mentions)
Lsrii flow cytometer, by BD (21 mentions)
Facsverse, by BD (20 mentions)
Facsaria 3, by BD (19 mentions)
1 194 protocols using 7 aad
1
Radiation-Induced Apoptosis Quantification
2
Annexin V Staining Apoptosis Analysis
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For Annexin V staining, cells with YM155 or YM155 analogs treated for 24 h, were washed twice with PBS and were stained with FITC-Annexin V (BD Bioscience, # 556419) and 7-AAD (BD Bioscience, # 559925) or PE-Annexin V (BD Bioscience, # 556421) and 7-AAD for 30 min at RT in the dark. Cell death was analyzed with FACS as described previously (BD Bioscience) (Kwon et al., 2017 (link)). Cells stained with Annexin / 7-AAD were analyzed by FACS Calibur I (BD Bioscience). For all of the cell images captured, Light channel of optical microscope (Olympus, CKX-41) or JULI-stage (NanoEntek) were used according to the manufacture's protocol.
3
Quantifying Apoptosis in Jurkat Cells
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An apoptosis detection kit with PE annexin V and 7-AAD (BioLegend, San Diego, CA, USA) was used to analyze the Jurkat cells to determine apoptotic cell percentages. Once the cells were stained with PE annexin V and 7-AAD, a flow cytometer (BD FACSVerse, BD Biosciences, San Diego, CA, USA) was used to classify the cells as early (7-AAD-/annexin V+) or late (7-AAD+/annexin V+) apoptotic cells. The cell percentages were acquired using FlowJo software (Version 10, BD Biosciences). The detailed method followed the manufacturer’s instructions, as described in our previous publication [29 (link)].
4
Annexin V-PE Apoptosis Assay for HSPCs
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Annexin V–PE apoptosis detection kit (BD Pharmingen) was used to detect early apoptotic cells. Human HSPCs were washed with D-PBS and resuspended in Binding Buffer solution at the concentration of 106 cells/ml and labeled following manufacturer’s protocol. 7-Amino-actinomycin D (7AAD; BD Pharmingen) was also added to the cells to distinguish among live (7AAD−, AnnexinV−), early apoptotic (7AAD−, AnnexinV+), late apoptotic (7AAD+, AnnexinV+), and necrotic (7AAD+, AnnexinV−) cells. Samples were analyzed by flow cytometry with FACS LSRII (BD Biosciences) within 1 hour, and results were analyzed by the Flow-Jo 8.3 software (Tristar).
5
Apoptosis Analysis via Flow Cytometry
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Cells were reverse transfected with either control or STYK1 siRNAs, seeded in 6-well plates, and the indicated drugs were added after 48 hours. The cells were ultimately harvested after 96 hours of incubation. After harvesting, the cells were washed, resuspended in Annexin V binding buffer (556454, BD Biosciences, Franklin Lakes, NJ, USA), and then stained with 5 µl APC-Annexin V (550475, BD Biosciences) and 5 µl 7-AAD (559925, BD Biosciences) according to the manufacturer’s instructions. The measurements were done by a FACScanto flow cytometer (BD Biosciences). FACS data were analyzed for percentages of viable cells (AnnexinV-/7-AAD-), early-apoptotic cells (AnnexinV + /7-AAD-), and late apoptotic cells (AnnexinV + /7-AAD + ) using BD FACSDiva 8.0.1.
6
Flow Cytometry Analysis of Flavonoid-Induced Cell Death
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Cell death was analyzed by flow-cytometry as described previously [25 (link)]. Regarding Annexin V/7-AAD staining, cells at 24 h after treatment of each flavonoid were washed twice with PBS and stained with FITC conjugated Annexin V antibody (BD Bioscience, Franklin Lakes, NJ, USA, #556419) and 7-AAD (BD Bioscience, #559925) for an additional 45–60 min at room temperature in the dark. Cells stained with Annexin V/7-AAD were analyzed by FACS Calibur or FACS Lyric (BD Bioscience). Concerning all of the bright field images captured, a Light channel optical microscope (Olympus, Tokyo, Japan, CKX-41) or JULI-stage (NanoEntek, Seoul, Korea) was used in accordance with the manufacture’s protocol. The activity of caspase-3 was analyzed using a colorimetric active caspase-3 assay kit (Sigma–Aldrich, #CASP3C) according to the manufacture’s protocol.
7
Quantification of Oncolytic Herpes Virus Infection in Myeloma Cells
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Analysis of in vitro infection and killing of myeloma cell lines and primary cells by GFP-expressing oHSV-1 was done at the indicated time points by staining control and infected cells with V450 annexin V (catalog no. 560506, Becton Dickinson) and 7-AAD (catalog no. 559925, BD Pharmingen) following the manufacturer’s protocol and quantitating GFP+ and early (annexin+/7-AAD−) and late apoptotic cells (annexin V+/7-AAD+) by flow cytometry. Data were analyzed using FlowJo 2.0 (Tree Star, Ashland, OR, USA). Alexa Fluor 647 mouse anti-human HVEM (CD270, catalog no. 56441, BD Pharmingen) and mouse anti-human NECTIN-1 (CD111)-phycoerythrin (PE) (catalog no. 130-103-833, Miltenyi Biotec) were used to determine HVEM and NECTIN-1 expression, respectively, and flow data were analyzed using Kaluza software (Beckman Coulter). To measure HVEM (CD270) expression on plasma cells, cells isolated from BM extracts were stained with anti-CD138 fluorescein isothiocyanate (FITC) (Becton Dickinson), CD38 V450 (Becton Dickinson), and CD270 allophycocyanin (APC) (Becton Dickinson) to distinguish MM cells (CD138+, CD38+) from all other BM non-MM cells and then analyzed by flow cytometry.
8
Isolation of Skeletal Muscle Cell Populations
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Isolation of skeletal muscle FAPs, MuSCs, and double-negative cells was performed according to a previously reported protocol (86 (link), 87 (link)) with modifications. Limb muscles were dissected and mechanically dissociated in DMEM containing 10% horse serum (Hyclone), collagenase II (800 U/mL; Worthington), and dispase (1.1 U/mL; Thermo Fisher Scientific) at 37°C for 60 minutes. Digested suspensions were subsequently triturated by sterilized syringe with 20G 1/2 needle (BD Biosciences) and washed with DMEM to harvest mononuclear cells. Mononuclear cells were stained with corresponding antibodies. All antibodies used in FACS analysis were listed in Supplemental Table 1 . To exclude dead cells, 7-aminoactinomycin D (7-AAD) (MilliporeSigma) was used. Stained cells were analyzed and 7-AAD–Lin–Vcam–Sca1+ (FAP), 7-AAD–Lin–Vcam+Sca1– (MuSC), and 7-AAD–Lin–Vcam–Sca1– (double-negative) cells were isolated using FACSAria III cell sorter (BD Biosciences) with 4-way purity precision. Isotype control density plots were used as a reference for positive gating. Fluorescence density plot and population hierarchy of the cells were analyzed using FACSDiva (BD Biosciences) or FlowJo software (BD Biosciences).
9
Multiparametric Flow Cytometry Analysis
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Cells were stained with antibodies as previously described.6 (link) The following antibodies were used for staining: Annexin V-FITC (Becton Dickinson, San Jose, CA, USA), 7-AAD (Becton Dickinson), anti-human HLA-ABC (clone: W6/32)-FITC and -PE (eBioscience, Santa Clara, CA, USA), anti-human HLA-A2 (clone: BB7.2)-PE (Becton Dickinson), anti-human HLA-A24 (clone: 22E1)-PE (Medical & Biological Laboratories, Nagoya, Japan), and anti-human CD274 (B7-H1) (clone: MIH1)-PE (eBioscience). Isotype-matched immunoglobulin served as a negative control, and staining was detected with an LSR II flow cytometer (Becton Dickinson).
Dead and/or apoptotic cells were excluded using Annexin-V and 7-AAD. The relative mean fluorescence intensity (rMFI) was calculated according to the formula: [(MFI with specific mAb − MFI with isotype mAb)/MFI with isotype mAb]/[(MFI with specific mAb of control treatment − MFI with isotype mAb of control treatment)/MFI with isotype mAb of control treatment].
Dead and/or apoptotic cells were excluded using Annexin-V and 7-AAD. The relative mean fluorescence intensity (rMFI) was calculated according to the formula: [(MFI with specific mAb − MFI with isotype mAb)/MFI with isotype mAb]/[(MFI with specific mAb of control treatment − MFI with isotype mAb of control treatment)/MFI with isotype mAb of control treatment].
10
Macrophage-mediated Cytotoxicity Assay
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THP-1 cells were seeded in 96 well culture plate (SPL, 30096) with 10 ng/mL PMA and 500 ng/mL ionomycin for 48 hours, with media changed the next day for 24 hours. After media change on differentiated THP-1 cell, Target cells, PK(15) cells and PK(15)-hIL10 which is transfected human IL -10 in the PK(15) cell line were labelled with CellTrace™ CFSE (Life technologies™, C34554) and seeded in a 96 well plate with THP-1-dereived-macropahges. THP-1-differentiated-macrophages were co-cultured with target cells for 24 hours with E:T ratio; 1:1, 2:1, 5:1, 10:1, and 20:1. Co-cultured cells were stained by 7-AAD (BD, 559925) in 100 μL of PBS. The cytotoxicity of the macrophage was analyzed with the FACS Calibur flow cytometer (Becton Dickinson) as the percentage of dead cells (CFSE+7-AAD+).
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