This in vitro imaging assay makes it possible to quantify the effect of different cell treatments or different genotypes upon microglial phagocytosis. Using two cell types that are relevant to neurodegenerative disease, we use dead neuroblastoma cells for the phagocytic cargo, which are prepared in a way that can be easily and cheaply scaled up by large high content imaging screens. In a class 2 biological safety cabinet, dissociate SH-SY5Ys by aspirating the medium.
Add 4 ml of cell dissociation buffer, and remove the buffer immediately so that less than 1 ml remains as a thin film coating the cells. Incubate for 2 to 3 minutes at 37 degrees Celsius and 5%carbon dioxide. Then at 10 ml of HBSS to the T-75 flask.
And pipette the SH-SY5Ys into a 15 ml conical centrifuge tube. Centrifuge at 400xG for 5 minutes. Aspirate the supernatant, and re-suspend the cells in 2 ml of phenol red-free HEPES buffered media, making sure to break up clumps before fixation.
Fix the cells by adding 2 ml of 4%power formaldehyde to the tube and incubating for 10 minutes at room temperature with occasional gentle agitation. Add 10 ml of HBSS to the tube. Then, centrifuge at 1200xG for 7 minutes.
Aspirate the supernatant, and re-suspend the cell pellet in 2 ml of phenol red-free HEPES buffered media. Count and remove 1 million HS-SY5Y cells into a 2 ml low protein-binding tube. Bring the total volume to 300 to 500 microliters with phenol red-free HEPES buffered media.
Then briefly warm the tube in a 37 degrees Celsius water bath. Reconstitute the pH-sensitive red fluorescent dye STP ester according to manufacturer's instructions. Then add 12.5 micrograms of dye per million SH-SY5Y cells.
Mix gently by flicking the tube. And incubate the tube at room temperature for 30 minutes, protected from light. Add 1 ml of HBSS and centrifuge at 1200xG for 7 minutes and 4 degrees Celsius.
Discard the supernatant. And wash with 2 ml of HBSS. Re-suspend the cell pellet in phenol red-free macrophage media to a concentration of 0.2 to 1.2 million cells per ml, so that 50 microliters contain 10, 000 to 60, 000 cells.
In a biological safety cabinet, prepare a solution of deep-red fluorescent cell permeant succinimidyl ester-reactive dye in macrophage media. Add Hoechst 33342, and warm the working solution to 37 degrees Celsius in a water bath. Aspirate the iPSC macrophage medium gently, by pipetting the cell supernatant with a multi-channel pipette into a sterile reservoir.
Add 70 microliters per well of the dye solution to the iPSC macrophages using a multi-channel pipette. Incubate for 1 hour at 37 degrees Celsius and 5%carbon dioxide. Repair experimental treatments in phenol red-free macrophage media.
After the incubation, aspirate the iPSC macrophage medium very gently with a multi-channel pipette, and add 100 microliters of HBSS per well. Immediately remove HBSS by gentle pipetting. Then add 100 microliters of phenol red-free macrophage media, plus experimental treatments in separate wells.
Incubate for 10 minutes to 1 hour at 37 degrees Celsius and 5%carbon dioxide. Use a multi-channel pipette to add 50 microliters of the labeled SH-SY5Ys per well from the sides of each well at the edge of the liquid. Then incubate at 37 degrees Celsius and 5%carbon dioxide for 3 to 5 hours.
After the phagocytosis incubation, gently aspirate cell supernatants by pipetting with a multi-channel pipette and discard. Wash once with 100 microliters of PBS. Then fix the cells by adding 100 microliters of 2%power formaldehyde and incubating for 15 minutes at room temperature.
Aspirate wells and add 100 microliters of PBS. Either proceed directly to imaging with the high content microscope, or cover with plate sealer and foil, and store the plate at 4 degrees Celsius, until required. Turn on the high-content imaging microscope, and open the image capture software.
Load the assay plate into the microscope by clicking on the LOAD"icon at the top of the screen. Select the SETUP"tab. In the dropdown menus on the top left box, select the appropriate plate type.
The autofocus option:two peak"The objective:40x Water, NA 1.1"Confocal"mode, and Binning of 1. Flush the 40x Water objective before use via the settings menu. In the channel selection box, use the plus icon to add the channels DAPI, Alexa 647, and Alexa 568.
Set these to measure at a single plane of one micrometer. Optimize time and power settings for the staining efficiency of the assay plate. Ensure the channels are not measured simultaneously, by clicking on channel sequence to separate out the channels.
Under navigation and define layout, select the wells of measurement, and select 9 to 12 fields per well. During setup, click on a representative field on the plate map. And check each measurement channel in turn to ensure that the staining is present, and that the images are focused by adjusting the channel offset.
To upload the data to a server for remote analysis, click on the online jobs"box and the relevant screen name. Save the assay protocol by clicking on the save"button, and click on the run experiment"tab at the top. Name the experiment plate, then click on start"A live cell time lives phagocytosis assay showed that with 10, 000 SH-SY5Ys per well, the number of phagocytose particles per cell increased linearly with time and was inhibited by approximately 50%by Cytochalasin D.With higher amounts of SH-SY5Ys per well, phagocytosis exhibited poor linearity, likely due to poor segmentation of iPSC, macrophages, and SH-SY5Ys in a more crowded field of view.
The following results were obtained by performing a fixed-cell high content imaging, including this representative image of phagocytosis. Increasing the amount of SH-SY5Ys resulted in a higher number of phagocytosed particles per cell. The phagocytosis assay was validated using several inhibitors of phagocytosis.
Cytochalasin D and Jasplakinolide significantly inhibited phagocytosis by 91 and 90 percent respectively. And Bafilomycin A1 significantly reduced phagocytosis by 31%when pre-incubated for 1 hour prior to phagocytosis. Addition of recombinant Annexin 5 significantly reduced phagocytosis by 30%when added to wells immediately before SH-SY5Y addition.
Fixed SH-SY5Ys we're confirmed to expose Phosphatidylserine using a fluorescent Annexin 5 probe. Whereas live SH-SY5Ys were negative for Annexin 5 staining. Several lengths of phagocytosis duration, from 1 to 5 hours, were tested using staggered addition of phagocytic cargo.
The overall length of this protocall can be shortened by preparing the phagocytic cargo and staining the iPSC macrophages in parallel. If you want to do this, work out the timings in advance, and use your incubations to prepare solutions for the future steps.
