Induction of apoptosis was assayed using APOSTRANDTM ELISA apoptosis detection kit (Enzo Life Sciences, United States). It is a highly sensitive assay that can detect apoptosis in as little as 500 cells. The assay is based on the sensitivity of DNA in apoptotic cells to formamide denaturation and the detection of the denatured DNA with a monoclonal antibody to single-stranded DNA. For the assay, cells were seeded in 96 well plates, fixed for 30 min, attached to wells by drying for 20 min, treated with formaldehyde for 10 min, denatured for 35 min, blocked, incubated with antibody for 30 min, washed, incubated with peroxide substrate and read at 405 nm, exactly as per the suggested protocol.
Apostrand elisa apoptosis detection kit
Manufactured by Enzo Life Sciences
Sourced in United States
The ApoStrand™ ELISA apoptosis detection kit is a laboratory equipment product that measures apoptosis, a form of programmed cell death. The kit utilizes an enzyme-linked immunosorbent assay (ELISA) format to detect and quantify apoptosis-specific DNA fragments in cell samples.
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10 protocols using apostrand elisa apoptosis detection kit
1
Sensitive Apoptosis Detection Assay
2
Apoptosis Detection via ELISA
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Detection of apoptotic cells was performed using ApoStrandTM ELISA apoptosis detection kit (Enzo Life Sciences Inc., NY, USA) according to manufacturer's instructions (Pomari et al., 2015[45 (link)]). The apoptotic positive control (single stranded DNA in PBS) was also included in the analysis. Data are expressed as the percentage of cells, comparing the optical density of the treated/CTRL cells with the optical density of the positive CTRL included in the kit.
3
Quantitative Analysis of Apoptotic Markers
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After appropriate treatments, floating and adherent cells (obtained from the medium and a PBS wash, or after trypsinization, respectively) were pooled and pelleted by centrifugation. Cell pellets were washed with PBS, lysed with RIPA buffer, and assayed by ELISA determinations for (i) active human Caspase-3 (CBA045, Merck Chemicals Ltd. Nottingham, UK), Beclin-1 protein expression (E98557Hu, USCN life sciences, Houston, TX, USA) and DNA damage assay (EpiQuik in situ DNA Damage Assay Kit; Epigentek, Farmingdale, NY, USA). All experiments were performed following the manufacturer’s protocols. Analyses were performed in triplicate and presented data represent mean ± standard error (SE) and consider three replicated experiments. APOSTRAND™ ELISA apoptosis detection kit (code BML-AK120-0001) and p62 ELISA kit (code ADI-900-212-0001) were purchased from Enzo Life Sciences, Inc. (Farmingdale, NY, USA). Beclin 1 ELISA (code SEJ557Hu) was purchased from 2BScientific (Heyford Park, UK). Human MAP1LC3B / LC3B ELISA Kit was purchased from LifeSpan BioSciences (Seattle, WA, USA).
4
Detecting Apoptosis via Denatured DNA
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As DNA in apoptotic cells is sensitive to formamide, denatured DNA was detected by a monoclonal antibody against single-stranded DNA using an ApoStrand™ ELISA apoptosis detection kit (Enzo Life Sciences, Plymouth Meeting, PA, USA) according to the manufacturer’s protocol. Briefly, 1.0 × 106 cells were cultured in a 96-well microplate and treated with different concentrations of MFKF-AP1β. After 12 h, cells were fixed and dried for attachment to the plate surface. Cells were then treated with formamide and heated at 56 °C for 30 min, followed by incubation with an antibody mixture for 30 min after blocking non-specific binding sites. After washing, peroxidase substrate was added to each well, and the absorbance was measured at 405 nm using an ELISA reader (Bio-Tek Instrument Co., WA, USA).
5
Apoptosis Quantification in MCF-7 Cells
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MCF-7 cells were treated with IC50 of MO and DOX for 48 hrs and ApoStrand™ ELISA apoptosis detection kit (Enzo Life Sciences, BML-AK120, Plymouth Meeting, PA, USA) was used to detect apoptosis in cells according to the manufacturer’s protocol. The ApoStrand™ ELISA is based on the sensitivity of DNA in apoptotic cells to formamide denaturation and the detection of the denatured DNA with an antibody to single-stranded DNA.
6
Cell Viability and Apoptosis Assay
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Cells were plated and cultured for 24–72 h prior to treatments and/or transfection, as described above (see Suppl. Table 1). Cell viability was determined by the MTT test, as previously described (Lee et al. 2005 (link)). Trypan blue exclusion was used as a test for necrosis. Detached cells in the medium and adherent cells were collected, diluted 1:2 with 0.4% trypan blue and automatically counted (Countess II FL, Thermo Fisher Scientific). Apoptosis was detected using the APOSTRAND™ ELISA Apoptosis Detection Kit (Enzo Life Sciences, Inc.; cat. # BML-AK120) according to the manufacturer’s protocol. The APOSTRAND™ ELISA is based on the sensitivity of DNA in apoptotic cells to formamide denaturation and the detection of the denatured DNA with a monoclonal antibody to single-stranded DNA (ssDNA). Treatments were performed in FBS-free medium without phenol red. FBS-free medium without phenol red or PBS were used as negative controls whereas ssDNA included in the APOSTRAND™ ELISA served as a positive control.
7
Detecting Apoptotic DNA with ELISA
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As DNA in cells showing apoptotic characteristics are sensitive to formamide, denatured DNA was detected using a monoclonal antibody against single-stranded (ss) DNA using an ApoStrand™ ELISA apoptosis detection kit (Enzo Life Sciences, Plymouth Meeting, PA, USA) according to the manufacturer’s instructions.
8
Quantitative Apoptosis Assay
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Apoptosis assay was performed by ApoStrand ELISA Apoptosis Detection Kit (Enzo Life Sciences) according to the manufacturer’s instructions.
9
Measuring Preadipocyte Apoptosis by ELISA
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Degree of apoptosis in preconfluent preadipocytes was measured using an APOSTRAND ELISA apoptosis detection kit (Enzo Life Sciences, Farmingdale, NY) according to the manufacturer's protocols and measured using a microplate reader (Multiskan FC; Thermo Fisher Scientific Inc.) at 405 nm. Apoptosis data from pre-adipocytes plated on vehicle or ATRA (0.2, 2, 20, and 200 nM) were normalized to positive control of single-stranded DNA (ELISA detect singlestranded DNA as a marker of apoptosis) and viable cell number.
10
Quantifying Apoptotic DNA by ELISA
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Amounts of single-stranded DNA in cells were determined using ApoStrand ELISA apoptosis detection kit (Enzo Life Sciences). DNA in apoptotic cells is sensitive to formamide denaturation, and denatured DNA was detected using a monoclonal antibody against single-stranded DNA. 1 × 105 cells were cultured in 96-well plates and subjected to vitexin treatment. The assay was performed according to manufacturer instructions, and absorbance was measured at 405 nm with an ELISA reader (Bio-Tek Instruments).
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