Phagocytosis Assay: Quantifying Macrophage Engulfment

Phagocytosis assay was performed as described previously [32 (link)]. Briefly, target C2C12 cell necrosis was induced by heating the cells for 10 minutes at 65 °C. C2C12 cells were stained with 1 µM CellTracker Deep Red Dye (ThermoFisher, Waltham, USA) and added to MΦs at 5:1 ratio (dead cell/MΦ). After 1-h co-culture, target cells were washed away extensively and MΦs were detached by EDTA. MΦs were labeled with Alexa Fluor 488-conjugated anti-F4/80 antibody (Invitrogen, Carlsbad, USA) for 20 min and the percentage of engulfing cells was determined on a Becton Dickinson FACSCalibur flow cytometer.

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Al-Zaeed N., Budai Z., Szondy Z, & Sarang Z. (2021). TAM kinase signaling is indispensable for proper skeletal muscle regeneration in mice. Cell Death & Disease, 12(6), 611.

Publication 2021

CellTracker Deep Red Dye Alexa Fluor 488-conjugated anti-F4/80 antibody FACSCalibur flow cytometer

Alexa fluor 488 Antibody Assay Cell necrosis Co culture Edta Phagocytosis

Corresponding Organization :

Other organizations : University of Debrecen

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Variable analysis

independent variables

Heating C2C12 cells for 10 minutes at 65 °C to induce target cell necrosis

dependent variables

Percentage of MΦs that have engulfed the target C2C12 cells

control variables

Co-culture of C2C12 cells (stained with CellTracker Deep Red Dye) and MΦs at a 5:1 ratio (dead cell/MΦ)
Co-culture duration of 1 hour
Labeling of MΦs with Alexa Fluor 488-conjugated anti-F4/80 antibody for 20 minutes
Measurement of engulfing cells using a Becton Dickinson FACSCalibur flow cytometer

controls

Positive control: Not specified
Negative control: Not specified

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