Inducing apoptosis using chemical treatment and acidic pH, and detecting it using the Annexin V flow cytometric assay

Abstract

Cell death is important in physiology, and can happen as a result of structural damage, or as a sequence of programmed cellular processes known as apoptosis. Pathogenic alterations in apoptosis occur in a number of diseases, including cancer, viral infections, autoimmune diseases, immunodeficiencies, and degenerative conditions. Developing accurate and reproducible laboratory methods for inducing and detecting apoptosis is vital for research into these conditions. A number of methods are employed to detect cell death, including DNA fragmentation, the TUNEL assay, and electron microscopy although each has its limitations. Flow cytometry allows for the distinction between live, early apoptotic, late apoptotic and necrotic cells. In this protocol we successfully induce apoptosis using chemical treatment and treatment with low pH in solid tumour cell lines, and have optimized detection using the Annexin V/PI apoptosis assay.

In vitro anticancer evaluation of Enceleamycin A and its underlying mechanism

“…6A, the scatter plot for control and treated cells was demonstrated with four distinct populations of unstained for viable cells, Annexin V-Alexa flour for early apoptotic cells, and Annexin V/propidium iodide dual stained for late apoptotic cells. 33 The early/late apoptosis ratio shown in Fig. 6B was 25.8%/2.45%, 49.7%/10.7%, 58.4%/14.85, and 60.65%/19.2% at the concentration 0, 2, 4, and 8 μg mL −1 respectively, after 48 hours of treatment with Enceleamycin A.…”

“…The apoptotic-like features in MDA-MB-231 cells after treatment with Enceleamycin A were estimated by Alexa Fluor® 488 Annexin V/Dead Cell Apoptosis Kit (ThermoFisher Scientific). 33 In brief, seeding of 3.5 × 10 5 cells per well of MDA-MB-231 cells was done in a 6-well plate and enable to grow overnight. The culture was treated with Enceleamycin A (2, 4, and 8 μg mL −1 ) for 48 h. After being washed with binding buffer (1×), the cells were stained for 30 min by Alexa Flour Annexin V and propidium iodide at 37 °C in the dark.…”

l-Asparaginase from Solanum lycopersicum as a Nutraceutical for Acute Lymphoblastic Leukemia

“…Annexin V (5 μL) and PI (10 μL) were added to the tubes, gently mixed, and incubated for 20 min at room temperature (RT) in the dark. Cells were analyzed by flow cytometry within an hour. − …”

Flow cytometry: Aspects and application in plant and biological science

“…can all cause death. Unique morphological characteristics of cells experiencing PCD include cell shrinkage, blebbing of the plasma membrane, nuclear condensation, and DNA fragmentation [156]. DNA fragmentation, alterations in caspase and mitochondrial activity, and phosphatidylserine (PS) detection are other methods for studying PCD.…”

“…Each of these methods has advantages and disadvantages [156]. The fact that flow cytometry is quick and can be done on basic analyzers gives it several benefits over other methods.…”