Detection and Isolation of Apoptotic Bodies to High Purity

Summary

A workflow using flow cytometry or differential centrifugation is developed to detect, quantify and isolate apoptotic bodies from an apoptotic sample to high purity.

Abstract

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%.

Introduction

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 morphology^2,3. Step 1 (plasma membrane blebbing) is characterized by the formation of balloon-like structures on the cell surface known as bl....

Protocol

1. Induction of Apoptosis

1. Centrifuge cell sample at 300 x g for 5 min and discard supernatant to remove any pre-existing cell debris.
   NOTE: When using adherent cells, seed cells in advance and wash with 1x phosphate-buffered solution (PBS) prior to apoptosis induction.
2. Determine cell number and collect cells.
   NOTE: Depending on the assay post-isolation, we recommend a starting cell number of at least 1 x 10⁷ cells.
3. Resuspend in complete media (respective medium con.......

Discussion

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.......

Materials

Name	Company	Catalog Number	Comments
Cells e.g. cultured human THP-1 monocytes (clone number: TIB-202)	ATCC	-
RPMI 1640 medium	Life Technologies	22400-089
Penicillin-streptomycin mixture	Life Technologies	15140122
FSC	Gibco	10099-141
1x PBS	-	-
Annexin V FITC	BD Bioscience	-
TO-PRO-3 iodide	Life Technologies	T3605	TO-PRO-3 may cause skin, eye and respiratory irration. Avoid direct contact.
10x Annexin V binding buffer	BD Bioscience	556454
EDTA	Sigma-Aldrich	1001710526
Centrifuge tube (15 mL)	Cellstar	188271
Microcentrifuge tube (1.5 mL)	Sarstedt	72.690.001
Tissue culture incubator (37 °C, 5% CO2)	-	-
Centrifuge	Beckman Coulter	392932
FACS ARIA III Flow cytometer, configured with two lasers for FITC and APC detection	BD Bioscience	-
FACS Canto II Flow cytometer, configured with two lasers for FITC and APC detection	BD Bioscience	-
FACS Diva 6.1.1 software	BD Bioscience	-
FlowJo 8.8.6 software	-	-
UV Stratalinker 1800	Stratagene	-