Phagocytosis was measured in our cybrids (n = 4 Euro/Non-DM, n = 5 Euro/DM, n = 3 [Afr + Asi]/Non-DM, n = 3 [Afr + Asi]/DM) using a variation of the methods described by Vo et al.38 (link) All experiments were performed in triplicate.
Culture of Cybrids With Fluorescent Beads—For each cybrid, cells were seeded at a density of 500,000 cells/well in 2 mL of standard culture media in 2 separate 6-well plates and then incubated overnight at 37 °C with 5% CO2. Media were replaced with 2 mL of a solution of 1 μm, fluorescently-tagged latex microbeads (Fluoresbrite® YG Microspheres 1.00 μm; Polysciences, Inc., Warrington, PA) diluted 1:3,000 in standard culture media. For each cybrid, 1 plate was incubated 48 h at 37 °C with 5% CO2 in 2% O2 while the other plate was incubated for 48 h at 37 °C with 5% CO2 in room-air.
After incubation, wells were briefly washed 3 times with 2 mL PBS-EDTA per wash and trypsinized. For each well, 2 mL of standard medium was added before pipetting each content into a separate 15 mL conical tube. Then, 1.5 mL of standard media was added to each well and then added to its respective 15 mL conical tube. Cell suspensions were then titrated with a 5 mL pipette to separate into single cells, strained through separate 35 μm nylon filters into separate 5 mL test tubes (Corning™ Falcon™ Test Tube with Cell Strainer Snap Cap; Corning Inc., Corning, NY), and centrifuged for 5 min at 1000 RPM. For each tube, media were carefully removed, and the pellet was resuspended in 50 μL of PBS-EDTA.
Flow Cytometry Analysis of Phagocytosis—Each sample was loaded into an ImageSteamX Mark II Imaging Flow Cytometer (Luminex Corp., Austin, TX), excited using a 488 nm laser, and imaged at 40 × magnification. 5,000 images were collected for each sample.
Imageset data were then analyzed using IDEAS software (Luminex Corp.) Briefly, images were first gated by object diameter to isolate images with single cells. Then, this subset was gated by fluorescence to determine the images containing microspheres. Finally, this smaller subset was gated to identify images where microspheres had been internalized. To do this, the software generates a “mask” of the cell area, “erodes” this area by a few pixels from the outer edge, and then determines if a fluorescent signal is located within the eroded mask. An internalization ratio was then calculated as follows: [Single Cells that Internalized Beads] / [Total Single Cells]. For each cybrid and condition, this ratio was then normalized to the ratio of its age-matched and haplogroup-matched, Non-DM cybrid cultured in room-air.
Culture of Cybrids With Fluorescent Beads—For each cybrid, cells were seeded at a density of 500,000 cells/well in 2 mL of standard culture media in 2 separate 6-well plates and then incubated overnight at 37 °C with 5% CO2. Media were replaced with 2 mL of a solution of 1 μm, fluorescently-tagged latex microbeads (Fluoresbrite® YG Microspheres 1.00 μm; Polysciences, Inc., Warrington, PA) diluted 1:3,000 in standard culture media. For each cybrid, 1 plate was incubated 48 h at 37 °C with 5% CO2 in 2% O2 while the other plate was incubated for 48 h at 37 °C with 5% CO2 in room-air.
After incubation, wells were briefly washed 3 times with 2 mL PBS-EDTA per wash and trypsinized. For each well, 2 mL of standard medium was added before pipetting each content into a separate 15 mL conical tube. Then, 1.5 mL of standard media was added to each well and then added to its respective 15 mL conical tube. Cell suspensions were then titrated with a 5 mL pipette to separate into single cells, strained through separate 35 μm nylon filters into separate 5 mL test tubes (Corning™ Falcon™ Test Tube with Cell Strainer Snap Cap; Corning Inc., Corning, NY), and centrifuged for 5 min at 1000 RPM. For each tube, media were carefully removed, and the pellet was resuspended in 50 μL of PBS-EDTA.
Flow Cytometry Analysis of Phagocytosis—Each sample was loaded into an ImageSteamX Mark II Imaging Flow Cytometer (Luminex Corp., Austin, TX), excited using a 488 nm laser, and imaged at 40 × magnification. 5,000 images were collected for each sample.
Imageset data were then analyzed using IDEAS software (Luminex Corp.) Briefly, images were first gated by object diameter to isolate images with single cells. Then, this subset was gated by fluorescence to determine the images containing microspheres. Finally, this smaller subset was gated to identify images where microspheres had been internalized. To do this, the software generates a “mask” of the cell area, “erodes” this area by a few pixels from the outer edge, and then determines if a fluorescent signal is located within the eroded mask. An internalization ratio was then calculated as follows: [Single Cells that Internalized Beads] / [Total Single Cells]. For each cybrid and condition, this ratio was then normalized to the ratio of its age-matched and haplogroup-matched, Non-DM cybrid cultured in room-air.
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