Method Article

Quantification of Efferocytosis by Single-cell Fluorescence Microscopy

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DOI:

10.3791/58149

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August 18th, 2018

 ,  ,  , 
1Department of Microbiology and Immunology and the Center for Human Immunology, University of Western Ontario, 2Institute of Infection and Global Health, University of Liverpool, 3Department of Respiratory Research, Aintree University Hospital NHS Foundation Trust

In This Article

Summary

Efferocytosis, the phagocytic removal of apoptotic cells, is required to maintain homeostasis and is facilitated by receptors and signaling pathways that allow for the recognition, engulfment, and internalization of apoptotic cells. Herein, we present a fluorescence microscopy protocol for the quantification of efferocytosis and the activity of efferocytic signaling pathways.

Abstract

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling and cellular processes that control efferocytosis. This quantification can be difficult to perform as apoptotic cells are often efferocytosed piecemeal, thus necessitating methods which can accurately delineate between the efferocytosed portion of an apoptotic target versus residual unengulfed cellular fragments. The approach outlined herein utilizes dual-labeling approaches to accurately quantify the dynamics of efferocytosis and efferocytic capacity of efferocytes such as macrophages. The cytosol of the apoptotic cell is labeled with a cell-tracking dye to enable monitoring of all apoptotic cell-derived materials, while surface biotinylation of the apoptotic cell allows for differentiation between internalized and non-internalized apoptotic cell fractions. The efferocytic capacity of efferocytes is determined by taking fluorescent images of live or fixed cells and quantifying the amount of bound versus internalized targets, as differentiated by streptavidin staining. This approach offers several advantages over methods such as flow cytometry, namely the accurate delineation of non-efferocytosed versus efferocytosed apoptotic cell fractions, the ability to measure efferocytic dynamics by live-cell microscopy, and the capacity to perform studies of cellular signaling in cells expressing fluorescently-labeled transgenes. Combined, the methods outlined in this protocol serve as the basis for a flexible experimental approach that can be used to accurately quantify efferocytic activity and interrogate cellular signaling pathways active during efferocytosis.

Introduction

Apoptosis, or programmed cell death, is a highly-regulated physiological process that occurs in most multicellular organisms and is crucial for their development and homeostasis1. In addition to being involved in normal cell turnover and embryonic development, apoptosis enables the elimination of infected or damaged cells from tissues and can be triggered in response to infection, inflammation, cancer, and also by medical interventions such as radiotherapy or steroids1. Apoptotic cells expose "eat-me" signals on their cell surface which are recognized by receptors on a range of professional and non-professional phagocytes, collectively referred to as "efferocytes". Engagement of these receptors induces the uptake and degradation of the apoptotic cell by the efferocyte through a process known as efferocytosis2,3. Phosphatidylserine is the best characterized eat-me signal driving efferocytosis. It is normally confined to the inner leaflet of the plasma membrane, with apoptosis activating a lipid scramblase which disrupts this membrane asymmetry, thus exposing phosphatidylserine on the cell surface4. Phosphatidylserine is found on the extracellular surface of some non-apoptotic cells, such as mature macrophages and activated platelets. However, these cells are not efferocytosed due to the presence of "don't eat me" signals, such as CD47, on their cell surface5,6,7. Exposed phosphatidylserine is recognized by an array of efferocytic receptors expressed by efferocytes. Binding of these receptors to phosphatidylserine, either directly or through the aid of opsonins, activates signaling pathways that promote the engulfment of the apoptotic cell into a membrane-bound vacuole termed the efferosome8,9,10,11,12. The efferosome fuses sequentially with endosomes and lysosomes, which deliver the molecular machinery necessary to acidify the efferosome and to degrade the apoptotic cell cargo13,14. Once degraded, the apoptotic cell-derived materials are trafficked to the recycling endosome — a process which limits immune responses to apoptotic cell-derived antigens, and which may allow for recovery of nutrients from the apoptotic cell13,15. A failure in efferocytosis results in impaired clearance of apoptotic cells; these uncleared cells eventually undergo secondary necrosis. Necrotic cells release pro-inflammatory cytosolic contents, pathogens, and autoantigens into the extracellular milieu, thus driving a range of infective, inflammatory and autoimmune diseases16,17. Together, apoptosis and efferocytosis facilitate the removal of dying and dead cells and allow for the maintenance of tissue homeostasis.

Investigating the molecular mechanisms underlying efferocytosis requires methods that provide a clear quantification of apoptotic cell uptake. This quantification is complicated by the fact that unlike other uptake mechanisms such as endocytosis and phagocytosis18,19, efferocytosis may not result in the engulfment of intact target cell, resulting in the piecemeal uptake of the apoptotic cell by the efferocyte20. The protocol described herein describes an in vitro efferocytosis assay that provides accurate delineation of the internalized versus non-internalized portions of individual apoptotic cells and can be combined with a variety of fixed-cell and live-cell microscopy approaches. Traditional phagocytosis assays add antibodies specific to the phagocytic target at the end of the experiment in order to label non-internalized targets, where as our method differs by labelling the apoptotic target with covalently-linked biotin21,22. While apoptotic cell specific antibodies can be used in this assay, the biotinylation approach allows for any protein-bearing target to be labeled and avoids potential issues with secondary antibody cross-reactivity if immunostaining is performed. Specifically, we outline the preparation of apoptotic Jurkat cells that have been dual-stained with both a cell tracking dye and biotin. The cell tracking dye allows for apoptotic cell-derived materials to be tracked during efferocytosis, whereas surface biotinylation allows for the discrimination of internalized from non-internalized portions of efferocytosed apoptotic cells. We also describe the culture and preparation of J774.2 and THP-1 cell lines for use as murine and human efferocytes, monocyte-derived M2 macrophages as an example of primary cell efferocytosis, and Jurkat cells for use as efferocytic targets. These methods can easily be applied to other cell lines or primary cells, to target cells undergoing any form of cell death (e.g. apoptosis, necrosis and necroptosis), and to micron-sized mimics which simulate apoptotic cells through lipid coatings or coating with ligands specific to an efferocytic receptor of interest.

The method outlined in this protocol has several advantages over the flow cytometry based methods commonly used in the field23,24. By directly imaging the phagocyte-apoptotic cell interaction, combined with clear labeling of both total and non-internalized apoptotic cell material, quantitative measures of efferocytosis can be made. Moreover, the use of pH-insensitive fluorophores limits confounding factors such as the suppression of FITC and GFP fluorescence at lysosomal pH that confounds some alternative methods25. Lastly, while not described in detail, these methods can be employed using efferocytes expressing fluorescently-labeled transgenes, or with post-fixation immunostaining, to allow for quantification of signaling molecule activity and monitoring of the cellular processes during efferocytosis.

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Protocol

Collection of blood from healthy volunteers was approved by the Health Science Research Ethics Board of the University of Western Ontario. Venipuncture was performed in accordance with the guidelines of the Tri-Council Policy Statement on human research.

1. Culture and Preparation of the THP-1 Monocyte Cell Line

  1. Culture THP-1 monocytes as a suspension culture in T25 flasks at 37 °C + 5% CO2. Cells should be grown in 5 mL of Roswell Park Memorial Institute 1640 (RPMI 1640) + 10% Fetal Bovine Serum (FBS).
  2. Each day suspend cells evenly throughout the growth media by gently shaking the flask, then immediately count cells with a hemocytometer. Cells should be passaged once cell density reaches 1 x 106 cells/mL:
    1. Pre-warm RPMI 1640 + 10% FBS in a 37 °C water bath.
    2. Transfer 2 x 105 cells into a 1.5 mL microcentrifuge tube or a 15 mL conical tube, and pellet cells by centrifuging at 500 x g at room temperature for 5 min.
    3. Remove the supernatant without disturbing the cell pellet and resuspend the pellet in 1 mL (1.5 mL microcentrifuge tube) or 5 mL (15 mL conical tube) of phosphate-buffered saline (PBS).
    4. Centrifuge the tube at 500 x g at room temperature for 5 min. Remove the PBS without disturbing the cell pellet.
    5. Resuspend pellet in 1 mL of fresh RPMI 1640 + 10% FBS.
    6. Into a new T25 flask place 4 mL of warmed media, and to this add the resuspended cells from 1.2.5. Culture in a 37 °C + 5% CO2 incubator until the cells require passaging (typically 3 days), or until cells are required for an experiment.
  3. For an experiment with THP-1-derived macrophages, remove the required number of cells from the flask and plate prior to passaging:
    1. Place the required number of 18 mm circular glass coverslips (#1.5 thickness) into the wells of a 12-well plate — typically 1 coverslip per condition and/or timepoint. Into each well aliquot 5 x 104 THP-1 monocytes. The number of cells added to each well can be altered, if required.
    2. Bring up the total volume of each cell-containing well to 1 mL using RPMI + 10% FBS warmed to 37 °C.
    3. Add 100 nM phorbol 12-myristate 13-acetate (PMA) to each well and culture for 3 days to induce differentiation of THP-1 monocytes into macrophage-like cells.

2. Culture and Preparation of the J774.2 Macrophage Cell Line

  1. Culture J774.2 cells in T25 flasks at 37 °C + 5% CO2. Cells should be grown in 5 mL of Dulbecco’s Modified Eagle Medium (DMEM) + 10% FBS and passaged once the culture reaches 80-90% confluency. To passage cells:
    1. Remove all media from the flask and rise once with 5 mL of PBS.
    2. Remove PBS from the flask and replace with 5 mL of fresh DMEM + 10% FBS.
    3. Using a cell scraper, scrape the bottom of the flask to suspend the cells in the media. Vigorously pipette the cells several times to break up any cell aggregates.
    4. Dilute cells 1:5 by removing 4 mL of the cell suspension from the flask and replacing it with 4 mL of fresh media. The remaining cell suspension can be discarded, used to start a new cell culture in a fresh T25 flask, or used for an experiment.
  2. To set up for an efferocytosis assay using J774.2 cells:
    1. One day prior to the start of the experiment, suspend J774.2 cells into 5 mL of fresh media, as per steps 2.1.1–2.1.3. Count cells using a hemocytometer and prepare the necessary volume of cells at a concentration of 5 x 104 cells/mL.
    2. Place the necessary number of 18 mm circular glass coverslips (#1.5 thickness) into the wells of a 12-well plate. Into each well aliquot 1 mL of the 5 x 104 cells/mL cell suspension.
    3. Culture overnight to allow the cells to adhere to the coverslip and recover from passaging.

3. Culture of Primary Human M2 Macrophages

  1. Collect 10 mL of heparinized human blood for every 12-well plate of M2 macrophages required.
  2. In a 15 mL centrifuge tube, layer 5 mL of human blood on top of 5 mL of pre-warmed Lympholyte-poly cell separation medium. Prepare multiple tubes if processing >5 mL of blood rather than using larger volume tubes.
  3. Centrifuge at 300 x g for 35 min, using medium acceleration and no break.
  4. Carefully remove the upper mononuclear-cell rich band using a plastic pipettor and transfer to a 50 mL centrifuge tube. If multiple tubes were prepared in step 3.2, the bands can be pooled into a single 50 mL tube. Bring volume of tube up to 50 mL with PBS.
  5. Centrifuge at 300 x g for 8 min and remove the supernatant. During this step place autoclaved 18 mm diameter circular coverslips (#1.5 thickness) into each well of a 12-well plate.
  6. Resuspend the cell pellet in 300 µL of serum-free RPMI 1640 per desired number of wells; e.g., if 10 mL of blood was processed to prepare a full 12 well plate, suspend cell pellet in 3.6 mL of media.
  7. Add 300 µL of the cell suspension to each coverslip-containing well in the 12-well plate. Incubate for 1 h at 37 °C + 5% CO2.
  8. Gently wash coverslip 3x with 1 mL of warmed PBS to remove any non-adherent cells.
  9. Add 1 mL of RPMI 1640 + 10% FBS + 10 ng/mL recombinant human M-CSF + cell culture antibiotic/antimycotic. Incubate at 37 °C + 5% CO2 for 5 days.
  10. Replace media with RPMI 1640 + 10% FBS + 10 ng/mL recombinant human M-CSF + 10 ng/mL recombinant human IL-4 + cell culture antibiotic/antimycotic. Incubate at 37 °C + 5% CO2 for 2 days to complete M2 polarization.
  11. Polarized macrophages should be used within the next 3 days.

4. Preparation of Apoptotic Jurkat Cells

  1. Culture Jurkat cells in 5 mL of RPMI 1640 + 10% FBS at 37 °C + 5% CO2. Jurkats are a suspension cell line and can be maintained by passaging 1:5 into fresh, pre-warmed media every 3–5 days.
  2. To prepare apoptotic cells, allow the Jurkat culture to grow to high density (4–5 days after passaging). Aliquot 1.5 mL into a 1.5 mL microcentrifuge tube and pellet cells by centrifugation at 500 x g for 5 min.
  3. Discard supernatant and re-suspend cell pellet in 1 mL of serum-free RPMI 1640 medium containing 1 µM staurosporine.
  4. Incubate 16 h at 37 °C + 5% CO2 to render cells apoptotic.
  5. If desired, confirm induction of apoptosis by staining with Annexin V:
    1. Aliquot 100 µL of staurosporine-treated Jurkat cell culture into a 1.5 mL microcentrifuge tube. Pellet cells by centrifugation at 500 x g for 5 min, discard supernatant, and re-suspend cell pellet in 100 µL of serum-free RPMI 1640 medium.
    2. Add 1 µL of fluorescein isothiocyanate (FITC)-conjugated Annexin V and incubate for 10 min at room temperature in the dark.
    3. Add 900 µL of PBS and transfer the entire volume to a single well of a 12-well plate containing an 18 mm circular glass coverslip (#1.5 thickness). Spin down in a centrifuge equipped with a plate adaptor at 200 x g for 1 min to force cells to adhere to the coverslip. Alternatively, cells can be placed into a chambered slide for imaging.
    4. Image using a fluorescence microscope.

5. Quantifying Efferocytic Uptake and Dynamics Using a Fixed Cell Efferocytosis Assay and Inside-out Staining

  1. Prepare THP-1, J774.2 or M2 human macrophages as described in Sections 1-3, respectively.
  2. The evening prior to the start of the experiment prepare apoptotic Jurkat cells as described in Section 4.
  3. Immediately prior to the experiment, count apoptotic cells using a hemocytometer. Transfer sufficient numbers of apoptotic cells into a 1.5 mL microcentrifuge tube – we usually add 5 x 105 cells/well, providing a target:efferocyte ratio of 10:1.
  4. Pellet Jurkat cells by centrifugation at 500 x g for 5 min and resuspend in 500 μL of PBS.
  5. During the centrifugation, aliquot 10 µL of DMSO into a new 1.5 mL microcentrifuge tube. Dissolve into the DMSO a minimal amount of N-hydroxysuccinimidobiotin (NHS-Biotin). 5-10 crystals (~0.005 mg) is sufficient.
  6. Transfer the 500 μL apoptotic cell/PBS suspension to the DMSO/NHS-biotin containing tube. Then dilute a cell tracking dye to the manufacturer’s recommended concentration into the apoptotic cell suspension. Make certain to select a cell tracking dye that does not overlap spectrally with FITC-Streptavidin (e.g. red or far-red cell tracking dye).
  7. Incubate suspension for 20 min at room temperature in the dark. Add an equal volume of RPMI 1640 + 10% FBS and incubate for 5 min at room temperature in the dark to quench any unreacted dye.
  8. Pellet cells by centrifugation at 500 x g for 5 min, discard supernatant, and re-suspend the stained apoptotic cells in 100 µL of RPMI 1640 + 10% FBS per well of macrophages.
  9. Add 100 µL of stained apoptotic cell suspension dropwise to each well of macrophages. Centrifuge 200 x g for 1 min in a centrifuge equipped with a plate adaptor to force contact between macrophages and apoptotic cells.
  10. Incubate plate for the desired period of time at 37 °C + 5% CO2 in a tissue culture incubator. For macrophages, efferocytosed material is usually first detectable after 20–30 min, and is completed after 120–180 min.
  11. At the desired time point(s) remove cells from the incubator. Wash cells twice with 1 mL of room temperature PBS to stop efferocytosis and remove non-efferocytosed apoptotic cells.
  12. Add FITC-conjugated streptavidin at a 1:1,000 dilution to each well and incubate for 20 min in the dark. This will label the exposed biotin on any non-efferocytosed apoptotic cell material.
    NOTE: If desired, cell nuclei can be stained during this step by addition of 1:20,000 dilution of Hoechst 33342 or 4',6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI).
  13. Wash cells 3 times with 1 mL PBS, gently shaking or rocking the samples for 5 min per rinse. Fix cells with 4% paraformaldehyde (PFA) in PBS for 20 min at room temperature. Rinse cells once with PBS to remove excess PFA.
  14. Mount coverslips on a slide for imaging and transfer to a fluorescence microscope. Capture z-stacks of a sufficient number of cells for accurate quantification — typically 10–30 cells per condition. Non-internalized apoptotic cell material will be apparent in the resulting image as cell tracking dye labeled material enveloped by FITC-streptavidin staining, while efferocytosed material forms discrete cell tracking dye puncta free of any streptavidin staining.
  15. Quantify efferocytosis in the resulting images via a variety of measures:
    1. Calculate the efferocytic index by determining the average number of discrete efferosomes (cell tracking dye+/streptavidin- puncta) per macrophage. Quantify only macrophages bound to an apoptotic cell or containing ≥1 visible efferosomes. Record macrophages bound to an apoptotic cell, but lacking discrete efferosomes, as having an efferocytic index of 0.
    2. Calculate efferocytic efficiency by measuring the fraction of macrophages that contain ≥1 efferosome.
    3. Calculate the rate of efferocytosis by imaging cells fixed and stained at multiple time-points. Only image macrophages bound to apoptotic cells, or containing visible efferosomes, with z-stacks captured of each cell. Once imaged, determine the rate of efferocytosis:
      1. Using the streptavidin staining as a guide, and the freehand or polygon selection tool in FIJI/ImageJ26 (or other image analysis software package), circle all of the cell tracking dye+/streptavidin- (e.g. internalized) material in a single plane of the z-stack. Measure the integrated intensity of the cell tracking dye this region. Individual measures for each efferosome (e.g. diameter, positioning relative to the cell border or nucleus, etc.)15,27 can easily be acquired simultaneously with these measurements, greatly increasing the data collected during analysis.
      2. On the same z-section, using the streptavidin staining as a guide and the freehand or polygon selection tool, circle all of the cell tracking dye+/streptavidin+ (e.g. non-internalized) material in a single plane of the z-stack. Only include staining from apoptotic cells in contact with the phagocyte. Measure the integrated intensity of this region.
      3. Repeat steps 4.2.3.1 to 4.2.3.2 for the remaining z-sections of the image. Sum the integrated intensity of the efferocytosed (Σeff) and non-efferocytosed (Σne) materials. The fraction of the apoptotic cell which has been efferocytosed can be calculated for the cell as:
        figure-protocol-1
      4. Quantify the fraction efferocytosed for all cells at all time points. Note that the fluorescent intensity of apoptotic cells/efferosomes can vary between images due to variations in the uptake of cell tracking dyes by individual apoptotic cells, and due to changes in image acquisition parameters such as exposure time. As such, only compare normalized values such as Fraction Efferocytosed, or intensity-independent values such as efferocytic index and efferocytic efficiency, between images, between experimental conditions, and between repeat experiments.
    4. To ensure the resulting dataset accurately reflects the variation in efferocytosis between cells, conduct these analyses on the maximum number of cells possible in each experiment.
      NOTE: Efferocytic efficiency and efferocytic index can be rapidly calculated (a few seconds/cell), and we typically aim to analyze at least 100 cells per experiment, repeating each experiment at least 3 times. Calculating the fraction of efferocytosed material, and efferosome-specific measures are more laborious, typically taking 2–3 min/cell for an experienced analyst. For these calculations we quantify a minimum of 15 cells per condition, per experiment.
    5. Record efferocytic index, fraction efferocytosed and individual efferosome data on a per-cell basis.
      NOTE: This allows for data analysis using single-cell approaches, thus allowing for inter-cell variations to be quantified. Population-level analyses can still be conducted on these datasets by averaging single-cell data acquired in individual experiments. Efferosome-specific measurements can be analyzed at the population level, single-cell level, and as ensembles (e.g. as populations of efferosomes independent of the cells containing them)15.

6. Live Cell Efferocytosis Assay Using Apoptotic Cells

  1. Prepare THP-1, J774.2 or M2 macrophages as described in steps 1-3, respectively. If required, cells should be transfected with transgenes or other genetic constructs at least 18 h prior to performing any experiments.
  2. The evening prior to the start of the experiment, prepare apoptotic Jurkat cells as described in Sections 4 and 5 with the following modifications:
    1. Dilute cells and induce apoptosis as per 4.1–4.3, and collect the required number of apoptotic cells as per 5.3–5.4.
    2. Add a cell tracking dye at the manufacturer’s recommended concentration to the apoptotic cell suspension. Incubate suspension for 20 min at room temperature in the dark. Add an equal volume of RPMI 1640 + 10% FBS and incubate for 5 min at room temperature in the dark to quench any unreacted dye. Do not add NHS-biotin for these experiments.
    3. Pellet cells by centrifugation at 500 x g for 5 min, discard supernatant, and re-suspend stained apoptotic cells in 100 µL of RPMI 1640 + 10% FBS per well of macrophages.
  3. If required, label the macrophages by removing culture media from each well and adding 500 µL of PBS containing the manufacturers recommended dilution of a cell tracking dye that does not overlap spectrally with the cell tracking dye added to the apoptotic cells or any fluorescent transgenes expressed by the macrophages. Incubate for 20 min at room temperature in the dark, then add an equal volume of DMEM + 10% FBS and incubate for 5 min at room temperature in the dark to quench any unreacted dye.
  4. Transfer the macrophage-containing coverslip to a Leiden chamber. Add 400 µL of DMEM + 10% FBS, followed by 100 µL of the labeled apoptotic cell suspension. Mix by gently pipetting media 2–3 times.
  5. Transfer the Leiden chamber to a heated and CO2-perfused chamber of a live cell fluorescent microscope.
    NOTE: For microscope setups that lack CO2 perfusion capabilities, use tissue culture medium buffered with 1 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) rather than sodium bicarbonate to maintain physiological pH while the culture is exposed to air.
  6. Capture a time-lapse series containing a white light (phase contrast or DIC) image, and images of all fluorescent labels:
    1. Use a 100X objective lens for experiments where resolving fine details is required (e.g. membrane dynamics of phagocyte-apoptotic cell interactions). Meanwhile, more general measures (rate of apoptotic cell uptake, interaction time between macrophages and apoptotic cells, etc.) are best imaged using a 60X or 63X objective.
    2. If available, use point-visiting to image multiple fields-of-view during a single acquisition, thus increasing the number of efferocytic events captured in a single experiment
    3. Because efferocytes often engulf small fragments of apoptotic cells, rather than intact cells, it is best to capture z-stacks. To minimize phototoxicity, capture z-sections separated by the focal depth of your microscopes objective (typically 0.5–1.0 µm), through the thickness of the cell (typically 8-10 µm for macrophages, e.g. 8–20 slices/cell). This ensures that all engulfment and trafficking events will be visible in at least one z-plane. Alternatively, use large (1–5 µm diameter) apoptotic cell mimics or apoptotic cells that undergo minimal fragmentation during efferocytosis (e.g. heat shocked neutrophils) instead without z-stacking. We have described the preparation of both mimics and apoptotic neutrophils previously28.
    4. To minimize photobleaching, use bright fluorophores and capture images using acquisition settings that minimize photobleaching — e.g. low-intensity excitation combined with high camera gain and short exposure times29. We strongly recommend using a microscope equipped with a high-sensitivity electromagnetic charge-coupled device (EM-CCD) camera or spinning-disk confocal, and a high-speed piezoelectric mechanical stage, for this form of imaging.
  7. The number of possible analysis methods that can be applied to these time-lapse videos is extensive and beyond our ability to review here. As some examples, use manual or automated tracking software to track the fusion, fission and movement of efferosomes within cells15, quantify efferosome fusion dynamics with endolysosomes by colocalization analysis in macrophages expressing compartment-specific fluorescent transgenes13, monitor uptake processes such as probing and cup formation30, determine the recruitment dynamics of signaling and trafficking proteins to the efferosome13, and/or quantify the degradative activity of efferosomes using apoptotic cells labeled with pH-sensitive, oxidant-sensitive or protease-activated fluorophores31.

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Results

Overnight culture of Jurkat cells with 1 µM staurosporine results in apoptosis of >95% of cells, which can be confirmed with Annexin V staining (Figure 1). Other cell types can be used for these experiments, although the concentration of staurosporine and the duration of staurosporine treatment will need to be optimized for each cell line. For reliable detection and quantification of efferocytosis, >80% of cells should be apoptotic prior to addin...

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Discussion

The methods outlined in this protocol enable the imaging and quantification of the dynamic efferocytic process, using both fixed-cell and live-cell approaches. These approaches offer several advantages over commonly employed flow cytometry-based methods23,24. The use of inside-out staining with fixed samples provides a more robust and accurate quantification of the rate and extent of efferocytosis — indeed, many flow cytometry-based methods simply label apo...

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Disclosures

The authors declare no conflict of interest.

Acknowledgements

This study was funded by Canadian Institutes of Health Research (CIHR) Operating Grant MOP-123419, Natural Sciences and Engineering Research Council of Canada Discovery Grant 418194, and an Ontario Ministry of Research and Innovation Early Research Award to BH. DGW contributed some of the images presented, to the optimization of the protocols and to the writing of the manuscript; he was funded by a pump-priming grant from the university of Liverpool. CY is funded by a Vanier Graduate Scholarship and CIHR MD/PhD Studentship. The funding agencies had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
RPMI 1640 MediaWisent3500-000-EL
DMEM MediaWisent319-005-CL
Fetal Bovine Serum (FBS)Wisent080-150
PBSWisent311-010-CL
18 mm circular glass coverslips #1.5 thicknessElectron Microscopy Sciences72290-08Size and shape of coverslip is not critical, but 18 mm fit into the wells of a standard 12-well plate which simplifies cell culture
StaurosporineCayman Chemical81590Dissolve in DMSO at 1 mM (1,000x stock solution)
Annexin V-Alexa 488ThermoFisherR37174
EZ-Link NHS-BiotinThermoFisher20217Store in a dessicator. Do not prepare a stock solution.
DMSOSigma-AldrichD2650
CellTrace FarRedThermoFisherC34572
CellTrace OrangeThermoFisherC34851
Hoescht 33342ThermoFisher62249
FITC-StreptavadinThermoFisherSA1001
Lympholyte-poly cell sepration mediumCedarlane LabsCL5071
Recombinant Human M-CSFPeprotech200-04
Recombinant Human IL-4Peprotech300-25
J774.2 Macrophage Cell LineSigma-Aldrich85011428-1VL
THP-1 Human Monocyte Cell LineATCCTIB-202
Jurkat T Cell LineATCCTIB-152

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Efferocytosis QuantificationApoptotic Cell UptakeDual labeling ApproachCell tracking DyeSurface BiotinylationStreptavidin StainingLive cell MicroscopyFluorescent Reporter GenesZ stack Analysis

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