Apoptotic cell death was evaluated using APC Annexin-V Apoptosis Detection Kit with PI (Thermo Fisher Scientific, Waltham, MA USA). Briefly, 1.5 × 104/mL SKO-007(J3) cells infected with lentivirus pLKO.1-shRNA-GAS6 or non-target shRNA were culture in 24-well plates for 72h. Cells were then stained using Annexin-V/APC and PI according to the manufacturer’s instruction. Cell populations were acquired using FACS Canto II flow cytometer (BD Biosciences, San Jose, California, USA). Flow cytometric analysis was performed using Flow Jo Flow Cytometric Analysis Software.
Apc annexin 5 apoptosis detection kit with pi
Manufactured by Thermo Fisher Scientific
Sourced in United States
The APC Annexin-V Apoptosis Detection Kit with PI is a laboratory tool used to detect and quantify apoptosis, a type of programmed cell death. The kit utilizes Annexin V, a protein that binds to phosphatidylserine, and the DNA-binding dye propidium iodide (PI) to identify cells undergoing early and late apoptosis.
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4 protocols using apc annexin 5 apoptosis detection kit with pi
1
Apoptosis Evaluation by Flow Cytometry
2
Evaluating Cell Proliferation and Apoptosis
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The methodology for the evaluation
of the growth of human MCF-7 breast carcinoma cells was previously
described, except that cells were grown for 96 h for IC50 determinations.66 (link) U-87 MG and OVCAR-3
proliferation assays were performed plating 104 cells/cm2 in a Multiwell 24. The day after, cells were treated with
compound15 , and DMSO was used as a control. Cells were
counted 48 h later with a Bürker counting chamber, after dilution
in TrypanBlue (#T6146 Sigma-Aldrich). IC50 was calculated
by using data from the dose–response curves after 48 h of drug
treatment using Graphpad Prism 7.0 software, as previously described.67 (link)Apoptotic cell death was evaluated using
the APC Annexin-V Apoptosis Detection Kit with PI (Thermo Fisher Scientific).
Briefly, 1.5 × 106/mL cells were cultured in 48-well
plates, untreated or treated with different concentrations of15 for 48 or 72 h. Cells were then stained using annexin-V/APC
and propidium iodide according to the manufacturer’s instructions.
Cell populations were acquired using a FACS Canto II flow cytometer
(BD Biosciences). Flow cytometric analysis was performed using Flow
Jo Flow Cytometric Analysis Software.
of the growth of human MCF-7 breast carcinoma cells was previously
described, except that cells were grown for 96 h for IC50 determinations.66 (link) U-87 MG and OVCAR-3
proliferation assays were performed plating 104 cells/cm2 in a Multiwell 24. The day after, cells were treated with
compound
counted 48 h later with a Bürker counting chamber, after dilution
in TrypanBlue (#T6146 Sigma-Aldrich). IC50 was calculated
by using data from the dose–response curves after 48 h of drug
treatment using Graphpad Prism 7.0 software, as previously described.67 (link)Apoptotic cell death was evaluated using
the APC Annexin-V Apoptosis Detection Kit with PI (Thermo Fisher Scientific).
Briefly, 1.5 × 106/mL cells were cultured in 48-well
plates, untreated or treated with different concentrations of
and propidium iodide according to the manufacturer’s instructions.
Cell populations were acquired using a FACS Canto II flow cytometer
(BD Biosciences). Flow cytometric analysis was performed using Flow
Jo Flow Cytometric Analysis Software.
3
Annexin-V Apoptosis Detection in SKO-007(J3) Cells
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Apoptotic cell death was evaluated using APC Annexin-V Apoptosis Detection Kit with PI (Thermo Fisher Scientific, Waltham, MA, USA). Briefly, 15 × 104 SKO-007(J3) were cultured in 24-well plates, untreated or treated with BMSC-CM for 72 h. Cells were then stained using Annexin-V/APC and PI according to the manufacturer’s instruction. Cell populations were acquired using FACS Canto II flow cytometer (BD Biosciences). Flow cytometric analysis was performed using Flow Jo Flow Cytometric Analysis Software.
4
Cell Cycle and Apoptosis Analysis of Compound Treatments
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For cell cycle phase analysis, HeLa cells were seeded in 12-well plates at a concentration of 2.0 × 104 cells/well and incubated at 37 °C for 17 h. Then, cells were treated with varying concentrations of test compounds and incubated for an additional 17 h. Next, cells were harvested and washed with PBS. Then, cells were stained with propidium iodide (PI) according to the Cycle Test Plus DNA Reagent kit protocol (BD Biosciences. San Jose, CA, USA). Cells were analyzed using a FACS Calibur instrument (BD Biosciences).
To analyze apoptosis, cells were seeded in 6-well plates and incubated at 37 °C for 17 h. Cells were then treated with varying concentrations of test compounds and incubated for an additional 48 h. The APC Annexin V apoptosis detection kit with PI (Thermo Fisher Scientific) was used to detect apoptosis. The staining was done according to the manufacturer’s protocol. Flow cytometric analysis was performed using CytoFLEX (Beckman Coulter, Brea, CA, USA) and measured using FlowJo software (BD Biosciences).
To analyze apoptosis, cells were seeded in 6-well plates and incubated at 37 °C for 17 h. Cells were then treated with varying concentrations of test compounds and incubated for an additional 48 h. The APC Annexin V apoptosis detection kit with PI (Thermo Fisher Scientific) was used to detect apoptosis. The staining was done according to the manufacturer’s protocol. Flow cytometric analysis was performed using CytoFLEX (Beckman Coulter, Brea, CA, USA) and measured using FlowJo software (BD Biosciences).
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