2.7. Efferocytosis assay

The protocol as stated in the paper was detailed:

As phagocytic cells, we used primary monocyte-derived macrophages, THP-1 cell-derived macrophages, both control and cells with manipulated expression ofM6P/IGF2Ras described above, and M6P/IGF2R−/− mouse fibroblasts with or without ectopically expressed human M6P/IGF2R.

Human macrophages were differentiated from blood monocytes, isolated from healthy donors via CD14-beads, during the 7-day culture with recombinant human M-CSF followed by 2 days in macrophage serum free medium. Alternatively, macrophages were differentiated from human monocytic cell lines (e.g. THP-1 cells).

To generate apoptotic cells, Jurkat T cells were labeled with CFSE (see below, Molecular Probes, Invitrogen, Carlsbad, CA) or alternatively,

with eFluor 670 (eBioscience, Thermo Fisher Scientific, Waltham, CA), and apoptosis was induced by SSP (200 ng/ml) for 16

or 9 h. Apoptotic T cells were then pretreated for 30 min with Plg (100 nmol/l), washed with PBS, and cultured with the phagocytic cells

at a 5:1 (T cell: phagocyte) ratio.

Optionally, apoptosis may be triggered in different cell types. The cells (e.g. Jurkat T cell line) should be at first fluorescently labelled (e.g. CFSE), and then apoptosis will be induced by various stimuli (e.g. treatments with staurosporine, cisplatin, UV light, FasL, starvation, or a forced cells’ detachment). During early phases of apoptosis, phosphatidyl serine is externalised onto the outer plasma membrane whereon it is open to the staining with fluorescently labelled Annexin-V; later, the plasma membrane integrity is lost allowing fluorescent dyes, such as DAPI, propidium iodide, 7AAD, to enter the cell. Thus, apoptotic cells will be discriminated by specific staining and flow cytometric or microscopic detections: The early apoptotic cells (annexin-V+) will be distinguished from late apoptotic/necrotic cells (annexin-V+ / PI/7AAD+). Appropriate markers and controls for each cell type will be used.

Efferocytosis proceeded for 2–4 h at 37◦C in the absence and presence of the indicated molecules (TA 5 mmol/l; mAbs MEM-238 and MEM-240 to M6P/IGF2R, mAbs 4Pg and 7Pg to Plg, and control mAb AFP-01, all at 5 𝜇g/ml). For the mAbs experiments, the cells were co-treated with 2% beriglobin to block Fc receptors.

Afterward, the phagocytes fed with the CFSE-labeled apoptotic cells were thoroughly washed from the non-uptaken apoptotic Jurkat T cells and then harvested by trypsinization. By this process, any bound apoptotic cell should be cleaved off from the phagocytes’ surface.

Flow cytometry was used to quantify the percentage of cells that phagocytosed apoptotic cells labeled with either CFSE or eFluor 670.

CFSE labeling

•   Wash cells 2x in PBS at RT and resuspend cell to final concentration 1x 107 cells/ml.

•   Pipette cells suspension to a fresh tube containing CFSE at final concentration 2 M (range from 0.02-20 M possible) while vortexing (use wrap tube in aluminum foil).

•   Slowly shake at 37oc incubator for 10 minutes.

•   Uptake of dye is stopped by addition of ice-cold Hank’s balanced salt solution (HBSS) supplemented with 20% FCS to at least 3x volume.

•    The cells were then washed twice, the second time in HBSS without FCS and finally resuspended at 5-10x 105cells/ml in medium.

•    Cells were then cultured overnight at 37oc to allow complete efflux of all CFSE not stably bound to intracellular proteins.

•    Second day (at least couple of hours after labeling) after washing twice in complete medium they can be used for assay.

Take care:   Always suck out all media during washing, leave just the pellet and resuspend immediately.

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