Quantification of Efferocytosis by Single-cell Fluorescence Microscopy

This method can be used to answer key questions in the efferocytosis field such as the role of signaling molecules in mediating the uptake of apoptotic cells. The main advantage of this technique is that it provides a clear quantification of apoptotic cell uptake including partial or piecemeal apoptotic cell uptake which isn't always apparent with other methods. Though this method can provide insight into efferocytosis by immune cells it can also be applied to other cell types such as epithelial or cancer cells.

Generally, individuals new to this method will struggle because of the difficulty in optimizing microscope acquisition settings. After preparing human macrophages and apoptotic Jurkat cells, use a hemocytometer to count the apoptotic cells. Then transfer a sufficient quantity of apoptotic cells to a 1.5 milliliter microcentrifuge tube.

Pellet the Jurkat cells via centrifugation at 500 times g for five minutes. Then resuspend the cells in 500 microliters of PBS. While the Jurkat cells are in the centrifuge, aliquot 10 microliters of DMSO into a new microcentrifuge tube.

Next, dissolve a minimal amount of NHS-Biotin in the DMSO. After this, transfer 500 microliters of the apoptotic cell, and PBS suspension to the tube containing DMSO and NHS-Biotin. Then, dilute an appropriate cell-tracking dye according to the manufacturer's instructions.

Incubate the cell suspension at room temperature for 20 minutes in the dark. Then, add an equal volume of RPMI 1640 with 10%FBS, and incubate the suspension at room temperature in the dark for an additional five minutes. After this, pellet the cells via centrifugation at 500 times g for five minutes.

Then discard the supernatant, and resuspend the stained cells in 100 microliters of RPMI 1640 with 10%FBS. Add 100 microliters of stained cell suspension drop wise to each well of macrophages. Then centrifuge the plate at 200 times g for one minute.

Incubate the plate at 37 degrees Celsius with 5%CO2 in a tissue culture incubator. After the appropriate incubation period, remove the cells from the incubator. Then wash the cells twice with one milliliter of PBS.

After this, add diluted FITC-conjugated streptavidin to each well. And incubate the plate for 20 minutes in the dark. Wash the cells three times with one milliliter of PBS, and gently shake the samples for five minutes after each rinse.

Then fix the cells with 4%paraformaldehyde in PBS for 20 minutes at room temperature. Rinse the cells once with PBS to remove any excess PFA. Mount coverslips on an imaging slide, and transfer the slide to a fluorescence microscope.

Capture Z stacks of a sufficient number of cells for quantification. It is critical to set the distance between said sections such that all engulfed apoptotic material is detected. The maximum distance between said sections should be no greater than the focal depth of your objective lens, typically 0.5 to one micrometers.

Load one of the Z stacks into the software using the Import feature. Then select Integrated density as a measurement option under the Set Measurements panel. Analysis is easiest to perform if both the streptavidin and cell tracking dye channels are displayed as an overlay.

Any object with streptavidin staining anywhere along a circumference should be considered streptavidin positive, and therefore, should not be quantified as streptavidin negative. Once the image is loaded, using the streptavidin and the Freehand, or polygon selection tool, circle all of the cell tracking dye positives streptavidin negative material in a single plane of the Z stack. Then, measure the integrated intensity of the cell tracking dye in the region.

In the same Z section use the streptavidin staining, and the Freehand or polygon selection tool to circle all of the cell tracking dye positive streptavidin positive material. Finally, measure the integrated intensity of this region. Using this procedure, more than 95%of Jurkat cells treated with Staurosporine went through apoptosis.

In most experiments, the fraction of apoptotic cells that are efferocytosed increased in a time-dependent fashion. When optimizing this protocol for different efferocytic cell types, or different apoptotic cell targets, it is important to test the range of apoptotic cell to efferocytic cell ratios. Usually ratios of 10 to one or 30 to one work the best.

This procedure can be easily modified to track signaling molecule activation, or intracellular trafficking by expressing fluorescent reporter genes in the efferocytic cells, thus allowing for these processes to be imaged during efferocytosis.