Apoptosis rate was assessed by flow cytometry with the Annexin V-APC/PI apoptosis detection kit (BD Biosciences, CA, USA). Briefly, NPCs were pretreated with different doses of Kin for 2h before TBHP (100 μM) addition for 24h. Then the treated NPCs were harvested, washed twice with PBS, and resuspended in 100 μl binding buffer mixed with 5 μl of Annexin V-APC and 5 μl of PI. After incubation at room temperature in the dark for 15 min, the cells were detected in a BD Accuri C6 Flow cytometer (BD Biosciences, CA, USA) and the data were analyzed using FlowJo software (BD Biosciences, CA, USA).
Annexin 5 apc pi apoptosis detection kit
Manufactured by BD
Sourced in United States
The Annexin V-APC/PI apoptosis detection kit is a laboratory product designed to detect and quantify apoptosis, a type of programmed cell death, using flow cytometry. The kit contains Annexin V conjugated to the fluorescent dye APC (allophycocyanin) and the DNA-binding dye propidium iodide (PI). The core function of this kit is to enable the identification and differentiation of viable, early apoptotic, late apoptotic, and necrotic cells within a sample.
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7 protocols using annexin 5 apc pi apoptosis detection kit
1
Apoptosis Evaluation of NPCs
2
Apoptosis Detection in Cultured Cells
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Cultured cells were harvested by trypsinization and washed with 1× PBS. For each sample, 1 × 106 cells were stained with the annexin V‐APC/PI apoptosis detection kit (BD Biosciences, San Jose, CA, USA) according to the manufacturer’s instructions, and then analyzed using fluorescence-activated cell sorting (FACS; BD FACSCanto II, BD Biosciences, CA, USA).
3
Apoptosis Detection by Flow Cytometry
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Apoptotic levels of involved cells were detected after drugs treatment by flow cytometry using an Annexin V-APC/PI Apoptosis Detection Kit (BD, USA). Cells were collected after different drug treatments, then the assays were carried out according to the manufacturer's protocol. Samples were processed immediately using a FACSCalibur flow cytometer (BD, USA). Data was further analyzed by FlowJo 10.4. 20,000 cells were recorded and analyzed in all conditions.
4
Evaluating Apoptosis and Cell Cycle in AML
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U937, KG1α, HL-60, THP-1 and TF1α cells were seeded at 1 × 105 cells/well in 6-well plates in serum-containing media; cells were cultured for 12 h before treatment. CP-EPS8-NLS was added at concentrations ranging from 0 to 175 μM and incubated at 37 °C and 5% CO2 for 24 h and 48 h respectively in KG1α. U937, KG1α, HL-60, THP-1 and TF1α cells were added at 0 or 70 μM CP-EPS8-NLS for 24 h. CP-EPS8-NLS treated AML cells were collected and washed by PBS, suspended in binding buffer according to the manufacturer’s protocol (BD, Annexin-V-APC & PI Apoptosis Detection Kit). Cells were analyzed with CellQuest software and each measurement was repeated three times to ensure reproducibility. For cell cycle analysis, AML cells were cultured for 12 h before treatment. CP-EPS8-NLS was added to a final concentration of 0 or 70 μM and incubated at 37 °C and 5% CO2 for 24 h. Cells were collected, washed with PBS, and suspended in RNase A and PI for 1 h in the dark. Samples were analyzed on a FACSCalibur Flow Cytometer (Becton Dickinson, New Jersey, USA).
5
Quantifying Apoptosis and Characterizing CESCs
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The Annexin V‐APC/PI apoptosis detection kit (BD Biosciences, California) was used to detect and assess the rate of apoptosis via flow cytometry. The cells were digested and collected with 0.25% trypsin‐EDTA, washed with PBS, resuspended in 100 μL of binding buffer and stained with 5 μL of annexin V‐APC and 5 μL of 7‐AAD. After the cells were incubated for 30 minutes at room temperature, the apoptosis rate of the cells was detected in a flow cytometer (BD Biosciences). FlowJo software was used to analyze the collected data. To identify CESCs, antibodies against CD44 (103,005, BioLegend, San Diego, California), CD90 (202,503, BioLegend), and CD45 (103,107, BioLegend) were used. CESCs were collected within three generations and washed three times with PBS. After the cells were incubated at room temperature for 30 minutes and washed with PBS, the percentage of CESCs was detected and analyzed using flow cytometry (BD FACSCalibur).
6
Apoptosis Detection and CESC Isolation
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The Annexin V-APC/PI apoptosis detection kit (BD Biosciences, CA, USA) was used to detect and assess the rate of apoptosis via ow cytometry. The cells were digested and collected with 0.25% Trypsin-EDTA, washed with PBS, resuspended in 100 μl of binding buffer and stained with 5 μl of Annexin V-APC and 5 μl of 7-AAD. After the cells were incubated for 30 minutes at room temperature, the apoptosis rate of the cells was detected in a ow cytometer (BD Biosciences, CA, USA). FlowJo software was used to analyze the collected data. To identify CESCs, antibodies against CD44 (103,005, Biolegend, San Diego, CA, USA), CD90 (202,503, Biolegend), and CD45 (103,107, Biolegend) were used. CESCs were collected within three generations and washed 3 times with PBS. After the cells were incubated at room temperature for 30 minutes and washed with PBS, the percentage of CESCs was detected and analyzed by ow cytometry (BD FACSCalibur, USA).
7
Apoptosis Analysis by Flow Cytometry
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Apoptosis was analysed by ow cytometry with Annexin V-APC/PI Apoptosis Detection Kit (BD, USA). Harvested, washed and resuspended the treated cells in 500μl of buffer, then combining with AV and PI in dark place for 15 min. Cells were analyzed by a FACS ow cytometer (BD Biosciences). This experiment was repeated in triplicate.
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