Apoptosis was detected by flow cytometry after staining the cells with Annexin‐V/propidium iodide (PI; Becton Dickinson). Apoptosis was quantified using CellQuest or FlowJo software with Annexin‐positive and Annexin‐ and PI‐double‐positive cells regarded as “apoptotic.” Apoptosis rate of untreated control cells was subtracted to determine “specific apoptosis.”
Annexin 5 propidium iodide
Manufactured by BD
Sourced in United States, China
Annexin V/propidium iodide (PI) is a fluorescent dye-based assay used to detect and quantify apoptosis (programmed cell death) in cells. Annexin V binds to phosphatidylserine, which is externalized on the cell surface during early apoptosis. Propidium iodide is a DNA-binding dye that can only enter cells with compromised cell membranes, which occurs during late apoptosis or necrosis. This assay allows for the differentiation of viable, early apoptotic, and late apoptotic/necrotic cell populations.
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Lab products found in correlation
Facscanto 2, by BD (4 mentions)
Facscalibur, by BD (4 mentions)
4 phenylbutyrate 4 pba, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Pi apoptosis detection kit, by BD (2 mentions)
Eclipse 80i, by Nikon (2 mentions)
Facscalibur flow cytometer, by BD (2 mentions)
Novocyte flow cytometer, by Agilent Technologies (2 mentions)
Novoexpress software, by Agilent Technologies (2 mentions)
Accuri c6 plus system, by BD (2 mentions)
Facsdiva software, by BD (2 mentions)
Cell counting kit 8 cck 8 kit, by Dojindo Laboratories (1 mentions)
Pi rnase staining buffer, by BD (1 mentions)
Flag tag (flag), by Cell Signaling Technology (1 mentions)
Ldh cytotoxicity assay kit, by Beyotime (1 mentions)
Poly 1 c lmw, by InvivoGen (1 mentions)
Kod plus neo polymerase, by Toyobo (1 mentions)
Taq master mix dye plus, by Vazyme (1 mentions)
2 3 cgamp, by InvivoGen (1 mentions)
Poly da dt, by InvivoGen (1 mentions)
46 protocols using annexin 5 propidium iodide
1
Annexin-V and PI Apoptosis Assay
2
Investigating Cellular Stress Response Mechanisms
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DMEM medium and RPMI 1640 medium were obtained from Hyclone (Hyclone Laboratories, Logan, Utah, USA). Fetal bovine serum (FBS) was obtained from Gibco (Grand Island, NY, USA). A cell counting kit-8 (CCK8) kit was purchased from Dojindo Laboratories (Kumamoto, Kyushu, Japan). An ROS assay kit (S0033), MTT Cell Proliferation and Cytotoxicity Assay Kit (C0009), LDH Cytotoxicity Assay Kit (C0017), and N-acetyl-L-cysteine (NAC, S0077) were obtained from Beyotime Institute of Biotechnology (Shanghai, China), while 4-phenylbutyrate (4-PBA) was from Sigma–Aldrich (St. Louis, MO, USA). PI/RNase Staining Buffer (550825) and Annexin V/propidium iodide (PI) were purchased from Becton Dickinson Company (BD; Franklin Lakes, NJ, USA). The antibodies against β-actin (5057), Cyclin A2 (91500), Cyclin B1 (12231), Cyclin E1 (20808), Geminin (52508), GPR78 (3177), PERK (5683), IRE1α (3294), PDI (3501), Ero1-Lα (3264), and FLAG (14793) were acquired from Cell Signaling Technology (Boston, MA, USA). The antibody against dsRed (ab185921) was acquired from Abcam (Abcam, Cambridge, Cambridgeshire, UK).
3
RNA Extraction and Molecular Analyses
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TRIpure Reagent for RNA extraction was obtained from Aidlab (Beijing, China). 2×Taq Master Mix (Dye plus) were from Vazyme Biotech Co.,Ltd (Nanjing, China). Poly I:C-LMW, 2’3’-cGAMP and poly dA:dT were bought from InvivoGen (Hong Kong, China). The Golden Star T6 Super PCR mix polymerase was from Tsingke (Nanjing, China) and the KOD plus neo polymerase was from Toyobo (Shanghai, China). Annexin V/propidium iodide (PI) were purchased from Becton Dickinson Company (BD; Franklin Lakes, NJ, USA).
The rabbit mAbs of HA (3724S), LC3I/II (3868), cleaved caspase3 (Asp175) (9664S), TBK1 (3504S), p-TBK1 (5483S), IRF3 (11904S), FLAG (14793), GFP (2956) and β-actin (5057) were acquired from Cell Signaling Technology (Boston, MA, USA). The phosphorylated-IRF3 (Ser396) rabbit mAb (MA5-14947), Goat anti-rabbit IgG (H+L) cross-adsorbed 488 (35553) were from ThermoFisher Scientific (Shanghai, China), The mCherry rabbit pAb (ab183628) and Goat anti-mouse IgG H&L Alexa Fluor®594 (ab150120) were acquired from Abcam (Cambridge, UK). The STING rabbit pAb (19851-1-AP), Bax rabbit pAb (50599-2-lg), Bcl2 mouse mAb (60178-1-lg), Atg5 rabbit pAb (10181-2-AP), Atg16L1 mouse mAb (67943-1-Ig), were all purchased from ProteinTech (Wuhan, China). The HA mouse mAb, GFP mouse mAb, HRP anti-mouse IgG (HS201), and HRP anti-rabbit IgG (HS101) were all acquired from Transgen Biotech (Beijing, China).
The rabbit mAbs of HA (3724S), LC3I/II (3868), cleaved caspase3 (Asp175) (9664S), TBK1 (3504S), p-TBK1 (5483S), IRF3 (11904S), FLAG (14793), GFP (2956) and β-actin (5057) were acquired from Cell Signaling Technology (Boston, MA, USA). The phosphorylated-IRF3 (Ser396) rabbit mAb (MA5-14947), Goat anti-rabbit IgG (H+L) cross-adsorbed 488 (35553) were from ThermoFisher Scientific (Shanghai, China), The mCherry rabbit pAb (ab183628) and Goat anti-mouse IgG H&L Alexa Fluor®594 (ab150120) were acquired from Abcam (Cambridge, UK). The STING rabbit pAb (19851-1-AP), Bax rabbit pAb (50599-2-lg), Bcl2 mouse mAb (60178-1-lg), Atg5 rabbit pAb (10181-2-AP), Atg16L1 mouse mAb (67943-1-Ig), were all purchased from ProteinTech (Wuhan, China). The HA mouse mAb, GFP mouse mAb, HRP anti-mouse IgG (HS201), and HRP anti-rabbit IgG (HS101) were all acquired from Transgen Biotech (Beijing, China).
4
Zearalenone and 4-phenylbutyrate Cytotoxicity
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Zearalenone (ZEA), and 4-phenylbutyrate (4-PBA), were purchased from Sigma–Aldrich (St. Louis, MO, USA); Fetal bovine serum (FBS) and DMEM/F-12 medium were obtained from Gibco (Grand Island, NY, USA); the cell counting kit-8 (CCK8) was purchased from Dojindo Laboratories (Kumamoto, Japan); the LDH release assay, ROS assay kit (S0033) and ATP assay kit were obtained from Beyotime Institute of Biotechnology (Shanghai, China); and the cell cycle assay caspase 3 activity assay kit and Annexin V/propidium iodide (PI) were purchased from Becton Dickinson Company (BD; Franklin Lakes, NJ, USA). Dorsomorphin dihydrochloride (HY-13418) was purchased from Medchem express (Shanghai, China).
5
Distinguishing Cell Viability States
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To distinguish healthy, apoptotic, and necrotic cells after the contact of the cell cultures with selected concentrations of precursors, staining with annexin V/propidium iodide (BD Biosciences, Canada) was used. The method of cell cultivation, sample preparation, and pre-cultivation was the same as in the cytotoxicity test. Precursors were tested in concentrations 0.25 and 10 mg mL−1 and combinations of An+APS and AnH+APS in concentrations 0.1 + 0.1 and 5 + 0.1 mg mL−1. After that, the precursors solutions were removed, and the remaining adherent cells were rinsed with a phosphate buffered saline (PBS), treated with trypsin and added to a previously removed, nonadherent cell population from the same treatment. Cells were resuspended in an annexin binding buffer and stained by annexin V–FITC in a concentration of 5 µg mL−1 and by propidium iodide in a concentration of 5 µg mL−1. After 15 min in the dark, cells were analyzed by a BD FACSCanto flow cytometer (BD Biosciences, Canada).
6
Apoptosis Induction by DAC and BZM
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Cells were treated with DAC alone, BZM alone, and DAC combined with BZM for 72 h. The cells were washed with PBS and then resuspended in 100 μl of binding buffer. Annexin-V/propidium iodide (PI; BD Pharmingen, San Diego, CA, USA) staining assays were conducted and analyzed by flow cytometry (FC500; Beckman Coulter, Brea, CA, USA). TUNEL staining was performed using an In Situ Cell Death Detection kit (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer’s instructions. The percentages of TUNEL+ cells from images of 10 randomly selected fields for each group were identified at a magnification of 200× (ECLIPSE80I; Nikon, Tokyo, Japan).
7
Macrophage-Mediated Clearance of Apoptotic Jurkat Cells
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Human Jurkat T cells (catalog no. ACC 282, DSMZ, Braunschweig, Germany) were treated with 0.5 µM staurosporine in serum-free RPMI 1640 for 3 h, washed extensively with RPMI 1640 medium containing human serum, then cocultured with human macrophages (1:10 macrophage to AC ratio) for 3 h. Cell death was routinely controlled and quantified by staining with annexin V/propidium iodide (Immunotools, Friesoythe, Germany) by FACS analysis (LSR Fortessa, BD Life Sciences, Heidelberg, Germany).
8
Evaluating Cell Apoptosis by Flow Cytometry
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Cell apoptosis was assessed by annexin V/propidium iodide (BD Biosciences, San Jose, CA, USA). Cells were harvested, washed with PBS, and resuspended in 1× binding buffer at a concentration of 1 × 106 cells/mL. A total of 100 μL solution was transferred to a new tube and added with 5 μL of APC Annexin V and 5 μL of propidium iodide. Cells were incubated at room temperature for 15 min in the dark and then analyzed by a FACScan flow cytometer.
9
Apoptosis Evaluation: In Vitro and In Vivo
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To assess apoptosis in vitro, 5×104 cells were seeded in each well of 24-well plate, and after 48 h, the cells were hydrated with PBS and then washed twice. Subsequently, staining was performed with Annexin V/propidium iodide (BD Biosciences, San Jose, CA, USA), and the cells were evaluated using a BD FACSCanto II (BD, Biosciences, USA) flow cytometer. To assess apoptosis in vivo, all specimens were fixed in 4% paraformaldehyde overnight at 4°C, permeabilized with 0.1% Triton X-100 and then incubated with TUNEL staining solution (Beyotime, Haimen, China) for 1 h according to the manufacturer’s protocol. TUNEL-positive cells were counted in five randomly selected fields of the slide under a microscope (Olympus).
10
Apoptosis induction by ACUPA-M-WOG, M-WOG and WOG
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The apoptosis-inducing activities of ACUPA-M-WOG, M-WOG and free WOG to LNCaP cells were tested on a flow cytometry. Briefly, LNCaP cells were cultured in 6-well plates for 24–48 h, and then the cells were exposed to DMEM culture containing ACUPA-M-WOG, M-WOG or free WOG for an added 48 h, respectively. Then, cells were digested with trypsin, washed by PBS and stained with Annexin V/Propidium Iodide (BD PharMingen, San Diego, CA, USA). Both early apoptotic and late apoptotic cells were counted by flow cytometry in cell apoptosis determinations. Annexin V+/PI− cells meant early apoptotic cells, while Annexin V+/PI+ cells meant late apoptotic cells.
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