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 blebs^4,5. Step 2 (apoptotic protrusion formation) includes the formation of long membrane protrusions such as apoptopodia, beaded-apoptopodia and microtubule spikes^6,7,8. Lastly, Step 3 (ApoBD formation) includes the fragmentation of the apoptotic protrusions and/or ApoCells to generate ApoBDs^6,9. Previous findings have suggested a role of ApoBDs in aiding apoptotic cell clearance and mediating intercellular communication. For example, it is proposed that the fragmentation of an ApoCell into ApoBDs may generate small 'bite-sized' pieces that could be easily removed by surrounding phagocytes^2,10,11. Furthermore, ApoBDs may harbor a series of biomolecules such as DNA, RNA and proteins, which may be trafficked to surrounding cells to facilitate cell-cell communication^12,13,14. To functionally investigate these processes, it is vital to confirm three key parameters including (i) validation of apoptosis induction and ApoBD formation, (ii) isolation of ApoBDs, and (iii) confirmation of ApoBD purity.

Previously, a number of methods including flow cytometry and electron microscopy have been used to study apoptosis and ApoBDs^15,16,17,18. However, ApoBD detection and quantification are often difficult or overlooked. For instance, the most routinely-used flow cytometry-based apoptosis assay employs annexin V (A5, a protein that binds the externalized 'eat-me' signal phosphatidylserine (PtdSer)) and nucleic acid stain propidium iodide (PI)¹⁹. However, by using this universal stain combination, analysis assumes that there are only three types of cell subsets (viable cells, ApoCells and necrotic cells) in the sample. Furthermore, though considered as "a gold standard" by many researchers for apoptosis, flow cytometry assays and subsequent data analysis often excludes ApoBDs through an initial gating step selecting FSC/SSC^{intermediate-high} events. Therefore, we have recently developed a novel flow cytometry assay using A5 and TO-PRO-3, another nucleic stain that can be selectively taken up by caspase 3/7-activated pannexin 1 (PANX1) channels^7,20. As caspase 3-induced PANX1 activation precedes PtdSer exposure at the early stage of apoptosis, TO-PRO-3 differentially stains apoptotic and necrotic cells. In addition, this approach combined with our novel gating strategy includes all acquired events during data analysis and as a result, six cell/particle subsets are identified, including: (i) viable cells (FSC/SSC^{intermediate/high}, A5^low, TO-PRO-3^low), (ii) A5^- early ApoCells (FSC/SSC^{intermediate/high}, A5^low, TO-PRO-3^intermediate), (iii) A5⁺ ApoCells (FSC/SSC^{intermediate/high}, A5^high, TO-PRO-3^intermediate), (iv) necrotic cells or late ApoCells (FSC/SSC^{intermediate/high}, A5^high, TO-PRO-3^high), (v) ApoBDs (FSC/SSC^low, A5^intermediate, TO-PRO-3^{low/intermediate}), and (vi) debris (FSC/SSC^low, A5^low, TO-PRO-3^low)²⁰. Our approach emphasizes the importance of analyzing all cells/particle subsets and, more importantly, the separation of ApoBDs from cells and debris²⁰. Thus, this approach demonstrates an efficient technique to validate the induction of apoptosis and ApoBD formation simultaneously.

Traditionally, ApoBDs have been isolated through a variety of differential centrifugation approaches whereby ApoBDs can be separated from cells or other extracellular vesicles based on density. However, such centrifugation methods are often limited by low ApoBD purity, lack of a quantification step to confirm sample purity, and/or inability to separate cell type-specific ApoBDs^17,21,22. Therefore, we recently developed two approaches, a FACS-based and a new differential centrifugation-based approach which can be coupled with our previously established flow cytometry method to validate the induction of apoptosis and sample purity²³. ApoBD isolation via our FACS-based approach can enrich ApoBDs to up to 99% purity, and can be coupled with a variety of cell type-specific antibodies to isolate ApoBDs from mixed cell populations, tissue samples and bodily fluids²³. Furthermore, our revised differential centrifugation approach demonstrates an efficient method to isolate ApoBDs to >90% purity²³.

In this paper, we describe in detail our experimental procedure to validate apoptosis induction, and to detect and quantify ApoBD formation. The ApoBD isolation workflows using FACS-based and differential centrifugation-based methods are also elaborated and compared. The representative data demonstrate that the described methodology provides an effective cutting-edge tool for future ApoBD studies.

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 containing 10% (vol/vol) fetal calf serum, 50 IU/mL penicillin, 50 µg/mL streptomycin mixture) for a final concentration of 1 x 10⁶ cells/mL.
4. Aliquot ~2 x 10⁶ cells per well of a 6 well plate.
5. To induce apoptosis, remove the plate lid and irradiate cells at 150 mJ/cm² using a UV irradiator. This should take approximately 30-60 s.
   NOTE: Prior to irradiation, ensure that ~0.5 x 10⁶ cells are retained for the 'Untreated' cell control.
   NOTE: Apoptosis can also be induced via other methods such as anti-Fas or serum starvation⁶.
6. Incubate at 37°C, 5% CO₂ for 2-8 hours, depending on the cell line.
7. Using a bench top light microscope, visualize cells to confirm the presence of apoptotic morphologies, such as blebbing, apoptopodia formation, and ApoBD formation.
   NOTE: 40X magnification is sufficient
8. Using a P1000 pipette, pipette and collect apoptotic samples.
9. Wash the plate with 1x PBS and combine with remaining sample to ensure maximum yield.
10. Collect ~1/10^th for the 'Whole Apoptotic Sample' (WAS) control.
11. Collect the 'Untreated' sample.
12. Centrifuge both the WAS and Untreated samples at 3,000 x g for 6 min.
13. Resuspend in 1 mL of 1x PBS and set aside on ice.
14. For ApoBD isolation, continue to either step 2 or 3 with the remaining apoptotic sample.

2. ApoBD Isolation via FACS

1. Centrifuge the entire sample at 3,000 x g for 6 min.
2. Remove the majority of the supernatant without disrupting the pellet.
3. Resuspend in a staining solution containing 1 mL of 1x A5 binding buffer, 75 µL of A5-FITC, and 2 µL of TO-PRO-3 per 1x10⁷ cells.
4. Incubate sample in the dark at room temperature for 10 min.
5. Add 1-2 mL of 1x A5 binding buffer and centrifuge sample at 3,000 x g for 6 min to remove excess stain.
   NOTE: For mixed cell populations or tissue samples, perform an antibody staining step using a combination of cell type-specific markers in 1x A5 binding buffer and incubate on ice for 20 min (or as per manufacturer's protocol) before centrifugation at 3,000 x g for 6 min.
6. Resuspend sample pellet in 3 mL of FACS buffer (1x PBS, 1x A5 binding buffer, 10% FSC, 2 mM EDTA) per 1x10⁷ cells.
7. Filter through a 70 µm cell strainer into a round-bottom, polypropylene (flow cytometry) tube and keep samples on ice and in the dark.
8. Turn on the FACS machine and perform standard set up using a 100 µm nozzle, perform the drop delay, and ensure a stable stream.
9. Load the sample and set acquisition speed to ~1000 events/s.
10. Adjust FSC, SSC, APC (TO-PRO-3) and FITC (A5) voltages and position events within the FACS plots to ensure populations can be clearly separated.
11. Record 20,000 events.
12. Set up a gating strategy as in part 4.
13. In the sort layout, add the final ApoBD gate as the desired sorting population.
14. Begin acquiring the sample and perform a test sort by collecting 5,000-10,000 ApoBDs into a new tube containing ~250 µL FACS buffer.
15. Perform a system back flush and load sorted ApoBDs.
16. Acquire and record test-sort ApoBDs.
17. Check that the ApoBD purity is ~99% by comparing FSC (y-axis) vs A5 (x-axis events).
    NOTE: A5 staining may reduce slightly when re-analyzing samples due to laser bleaching.
18. Once high purity is achieved, load original sample and continue sorting until the desired number of ApoBDs has been obtained.
    NOTE: If necessary, dual sorting can be performed to simultaneously isolate ApoCells and ApoBDs.
    NOTE: When sorting over a long period of time, we recommend incubating the collection tube at 4 °C.
19. Once sorting is complete, collect a small portion of post-sort ApoBDs, post-sort ApoCells, Untreated, and WAS to validate apoptosis and confirm post-sort purity.
    NOTE: Although the test-sort and post-sort purity should not differ significantly, this is based on the stream settings and stability.

3. ApoBD Isolation via Differential Centrifugation

1. Centrifuge the remaining apoptotic sample at 300 x g for 10 min.
2. Collect the supernatant, leaving ~500 µL to avoid disrupting the cell pellet, and add into a new 15 mL conical tube.
3. Remove the remaining 500 µL and resuspend the cell pellet in 2 mL of 1x PBS (this represents the 'ApoCell-enriched fraction'.
4. Centrifuge the collected supernatant for 20 min at 3,000 g.
5. Check for a pellet and carefully remove the supernatant (the supernatant may contain small extracellular vesicles including microvesicles and exosomes).
6. Resuspend the pellet in 1 mL of 1x PBS (this represents the 'ApoBD-enriched' fraction)
7. Collect 100 µL of each Viable, WAS, ApoCell-enriched, and ApoBD-enriched samples in a new round-bottom, polystyrene (flow cytometry) tube.
8. Add 100 µL of stain containing 2x A5 binding buffer, 1:100 A5-FITC, and 1:1,000 TO-PRO-3
9. Incubate at room temperature for 10 min in the dark.
10. Analyze samples by flow cytometry, using the gating strategy as described above to validate the successful induction of apoptosis and purity of the ApoBD-enriched fraction.

4. Flow Cytometry Gating Strategy

1. Plot TO-PRO-3 (y-axis) against FSC (x-axis) to separate necrotic cells (TO-PRO-3^high) from all other non-permeabilized events (TO-PRO-3^{low/intermediate}).
2. Select all non-permeabilized events and plot SSC against A5. Gate two populations including population 1 (P1), SSC^{intermediate/high}, A5^{low/intermediate} cells and population 2 (P2), all other events.
3. From P2, plot TO-PRO-3 against A5 and select A5^{intermediate/high} events to exclude all cell debris.
4. Select all A5 positive events and plot FSC against A5. Separate ApoBDs (FSC^low) from ApoCells (FSC^{intermediate/high}).
   NOTE: When gating ApoBDs for ApoBD isolation via the FACS-based approach, we recommend a final step by selecting ApoBDs and comparing TO-PRO-3 to A5 and selecting all events. This ensures that the final sorting gate uses fluorescence rather than FSC/SSC parameters.
5. For viable cell analysis, select P1 and perform one of two gating strategies. For general viable cell analysis, plot FSC against A5 and select all FSC^{intermediate/high} cells, therefore removing remaining cell debris. Alternatively, for in-depth analysis, viable cells can be separated from A5^- early ApoCells by plotting TO-PRO-3 against FSC. Select TO-PRO-3^low, FSC^{intermediate/high} viable cells and TO-PRO-3^intermediate, FSC^{intermediate/high} A5^- early ApoCells.

Results

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...

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

List of materials used in this article

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	-