Fixed Cell Efferocytosis Assay: A Method to Study Efferocytic Uptake of Apoptotic Cells by Macrophages Using Fluorescence Microscopy

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human welfare and have been reviewed by the local institutional review board.

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 the 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 time point. 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 1.1.1–1.1.3. 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 the 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 the 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/ImageJ (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.) 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 (Σ_eff) and non-efferocytosed (Σ_ne) materials. The fraction of the apoptotic cell which has been efferocytosed can be calculated for the cell as:
          
       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).