Published: August 12th, 2018
A workflow using flow cytometry or differential centrifugation is developed to detect, quantify and isolate apoptotic bodies from an apoptotic sample to high purity.
Apoptotic bodies (ApoBDs), microvesicles and exosomes are the key members of the extracellular vesicle family, with ApoBDs being one of the largest type. It has been proposed that ApoBDs can aid cell clearance as well as intercellular communication through trafficking biomolecules. Conventional approaches used for the identification and isolation of ApoBDs are often limited by the lack of accurate quantification and low sample purity. Here, we describe a workflow to confirm the induction of apoptosis, validate ApoBD formation, and isolate ApoBDs to high purity. We will also outline and compare fluorescence-activated cell sorting (FACS) and differential centrifugation based approaches to isolate ApoBDs. Furthermore, the purity of isolated ApoBDs will be confirmed using a previously establish flow cytometry-based staining and analytical method. Taken together, using the described approach, THP-1 monocyte apoptosis and apoptotic cell disassembly was induced and validated, and ApoBD generated from THP-1 monocytes were isolated to a purity of 97-99%.
Apoptosis, a well-studied form of programmed cell death, is required to maintain physiological homeostasis and remove potentially harmful cells within the human body1. After the induction of apoptosis, apoptotic cells (ApoCells) can undergo a series of morphological changes and disassemble into small membrane-bound vesicles termed ApoBDs. Overall, this process is known as apoptotic cell disassembly and can be divided into 3 distinct steps based on morphology2,3. Step 1 (plasma membrane blebbing) is characterized by the formation of balloon-like structures on the cell surface known as bl....
1. Induction of Apoptosis
Using the procedure outlined here, THP-1 monocyte apoptosis was induced and ApoBDs were detected and isolated via either a FACS-based or a differential centrifugation approach (Figure 1). Firstly, apoptosis was induced by UV irradiation and samples were collected after 2-3 h of incubation when cells exhibited apoptotic morphologies, including blebbing, apoptotic membrane protrusion formation and the generation of ApoBDs6. A TO-PRO-3 an.......
Since its early description in the 1950s, the field of apoptosis has advanced markedly, becoming a prominent research area. Despite the broad interest and extensive efforts, certain aspects of apoptosis, in particular the formation of ApoBDs, have not been well studied due to the lack of appropriate methodologies. These notably include the limitation in tracking apoptosis progression and ApoBD formation simultaneously using the traditional flow cytometry A5/PI analysis and the impurities of ApoBD isolation. We have recen.......
|Cells e.g. cultured human THP-1 monocytes (clone number: TIB-202)
|RPMI 1640 medium
|Annexin V FITC
|TO-PRO-3 may cause skin, eye and respiratory irration. Avoid direct contact.
|10x Annexin V binding buffer
|Centrifuge tube (15 mL)
|Microcentrifuge tube (1.5 mL)
|Tissue culture incubator (37 °C, 5% CO2)
|FACS ARIA III Flow cytometer, configured with two lasers for FITC and APC detection
|FACS Canto II Flow cytometer, configured with two lasers for FITC and APC detection
|FACS Diva 6.1.1 software
|FlowJo 8.8.6 software
|UV Stratalinker 1800
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