Apoptosis was analyzed by flow cytometry using the double staining with lipophilic Annexin V and 7-amino actinomycin D (7-AAD) (Abcam, Cambridge, United Kingdom), as described elsewhere (Wang et al., 2017 (link)). Briefly, after treatment, cells were harvested and washed with FACS buffer. Thereafter, cells were resuspended in 100 μl binding buffer (eBioscience) with 2.5 μl Annexin V (eBioscience) and incubated for 20 min at room temperature. 7-AAD (5 μl) was added 5 min before analysis. Early apoptotic cells (Annexin V-positive, 7-AAD-negative), necrotic/late apoptotic cells (double-positive), and living cells (double-negative) were determined by flow cytometry (BD LSR Fortsessa, BD Bioscience, Franklin Lakes, NJ, United States).
7 amino actinomycin d 7 aad
Manufactured by Abcam
Sourced in United Kingdom
7-amino actinomycin D (7-AAD) is a fluorescent dye used for identifying dead cells in a cell population. It binds to DNA and emits red fluorescence upon excitation.
Automatically generated - may contain errors
Lab products found in correlation
7 aad, by BD (2 mentions)
Annexin 5, by Abcam (2 mentions)
Lsr fortsessa, by BD (2 mentions)
Binding buffer, by Thermo Fisher Scientific (1 mentions)
Annexin 5, by Thermo Fisher Scientific (1 mentions)
Annexin 5, by BD (1 mentions)
High performance liquid chromatography grade acetonitrile, by Merck Group (1 mentions)
Phosphate buffered saline (pbs), by Corning (1 mentions)
Horse serum, by Thermo Fisher Scientific (1 mentions)
Istradefylline, by MedChemExpress (1 mentions)
Staining buffer, by Thermo Fisher Scientific (1 mentions)
Multifunctional microplate reader, by Tecan (1 mentions)
Luciferin luciferase based atp assay kit, by Beyotime (1 mentions)
Hmgb1 elisa kit, by Shino-Test (1 mentions)
4 protocols using 7 amino actinomycin d 7 aad
1
Apoptosis Analysis by Flow Cytometry
2
Annexin V-FITC/7-AAD Apoptosis Assay
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Apoptosis was analyzed by using the double staining with the lipophilic dye Annexin V and 7-amino actinomycin D (7-AAD) (Abcam, Cambridge, UK). Briefly, 0.5 × 106 cells were treated with AF for 2 h, harvested by trypsin, washed twice with PBS and resuspended in 100 μl. Thereafter, 2.5 μl Annexin V-FITC and 5 μl 7-AAD were added to the cell suspensions and incubated for 20 min at room temperature in the dark. Early apoptotic cells (Annexin V-positive, 7-AAD-negative), necrotic/late apoptotic cells (double-positive), and living cells (double-negative) were determined by flow cytometry (BD LSR Fortsessa, BD Bioscience, Franklin Lakes, USA).
3
Astragalus-based Natural Product Assay Protocol
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AR was obtained from Shanghai Guoda Drugstore Co., Ltd. (Shanghai, China). Isoastragaloside I and astragaloside IV were obtained from Shanghai Yuanye Biotech Co., Ltd. (Shanghai, China). Istradefylline was purchased from MedChemExpress Co., Ltd. (Princeton, NJ, USA). Ebselen was purchased from Shanghai Macklin Biochemical Co., Ltd. (Shanghai, China). The NK-92MI and K562 cell lines were purchased from the Cell Bank of the Shanghai Branch of the Chinese Academy of Sciences (Shanghai, China). Minimum essential medium α (α-MEM), fetal bovine serum (FBS), and horse serum were purchased from Gibco Life Technology Co., Ltd. (Logan, UT, USA). 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) was purchased from BioGems International, Inc. (Rocky Hill, NJ, USA). 7-aminoactinomycin D (7-AAD) was purchased from Abcam Co., Ltd. (Cambridge, UK). The lactate dehydrogenase (LDH) assay kit was purchased from Dojindo Chemical Technology Co., Ltd. (Shanghai, China). Phosphate buffered saline (PBS) was purchased from Corning Inc. (Corning, NY, USA). Silica gel (5 μm, 200 Å) was purchased from Meigao Chemical Co., Ltd. (Qingdao, China). High performance liquid chromatography (HPLC)-grade acetonitrile and mass spectrometry (MS)-grade ammonia acetate were obtained from Sigma-Aldrich Co., Ltd. (St. Louis, MO, USA).
4
Quantifying ATP, HMGB1, and Calreticulin in Cell Supernatants
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Cell supernatants were harvested at 3h, 6h, 12h, and 24h after the corresponding intervention in the above experiments in vitro. The ATP concentration was quantified by the luciferin/luciferase-based ATP assay kit (Beyotime Biotechnology Co., Ltd., China). Bioluminescence was assessed by a multifunctional microplate reader (Tecan, Austria). The high-mobility group box B1 (HMGB1) level in the supernatant was determined by an HMGB1 ELISA Kit (Shino-Test Corporation, Japan). For calreticulin (CRT) detection, 1×106 cells were harvested at 24h after the corresponding intervention and resuspended in 100μL of staining buffer (eBioscience, CA, USA). Prepared cells were incubated for 30min with anti-CRT (Alexa Fluor® 647) antibodies at 4°C in the dark. The samples were then washed and incubated with the viability detection/exclusion dye 7-aminoactinomycin D (7AAD) (Abcam, UK). Then, 7AAD-negative cells (dying cells) were gated, and membrane CRT rather than total CRT was detected. The mean fluorescent intensity (MFI) of CRT was calculated using FlowJo 10.0 software.
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