This study investigates the interaction between mesenchymal stem cells (MSCs) and macrophages, focusing on the enhancement of macrophage phagocytosis through MSC-mediated mechanisms. The use of zymosan conjugated to a pH-sensitive fluorophore allows for the measurement of phagocytosis in co-culture systems.
Take a multiwell plate with mesenchymal stem cells, or MSCs, in selected wells.
Introduce macrophages and incubate, facilitating MSC-macrophage interaction in the co-culture wells.
Add zymosan — a yeast cell wall component — conjugated to a pH-sensitive fluorophore to all the wells.
Macrophage scavenger receptors bind to zymosan, triggering its engulfment inside a phagosome, which fuses with a lysosome to form a phagolysosome.
An acidic pH inside the phagolysosome causes the fluorophore to emit fluorescence.
In the co-culture wells, MSCs project tunneling nanotubes, or TNT, cytoplasmic extensions that bridge with macrophages. MSC mitochondria travel through the TNT to enter the macrophages, increasing the total number of functional mitochondria.
An elevated mitochondrial number augments the production of adenosine triphosphate or ATP, which fuels the energy requirement for an increase in phagocytosis.
Record the fluorescence intensity over time from the internalized fluorophore-conjugated zymosans.
Co-culture shows increased fluorescence compared to wells with only macrophages, indicating MSC-mediated enhancement of macrophage phagocytosis.
Gently aspirate the medium from the experimental wells containing mesenchymal stem cells. Use a multichannel micro-pipette to add 100 microliters of the treated and the control macrophage cell suspensions in the appropriate experimental wells according to the plate design, and incubate overnight as demonstrated. Add one milliliter of live-cell imaging medium to the vial containing one milligram of zymosan particles, and collect the medium particle mixture into a glass culture tube.
After rinsing the vial with an additional one milliliter of imaging solution, transfer the rinsed imaging medium in the glass tube. Then, add 3 milliliters of additional imaging solution to achieve 0.2 mg/ml zymosan particle suspension. Vortex the zymosan suspension using quick pulses for 30 to 60 seconds. Then, use a probe sonicator to sonicate the suspension with 60 quick pulses. After aspirating the medium, rinse the experimental wells in reagent blank wells with 100 microliters of live-cell imaging solution.
Next, aspirate the live-cell imaging solution from the wells, and add 100 microliters of the zymosan suspension. Open the plate tray of the fluorescent reader using the touch-pad interface. Set the plate without the lid in the tray with the A1 well, in the upper left corner. Close the tray using the touch-pad, and click on the green "Read" button in the top menu.
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