Celltracker red dye

Cell lines were stained with CellTracker™ Green dye (Invitrogen) or CellTracker™ Red dye (Invitrogen) for 30 minutes at 37℃ in the absence of serum and washed with PBS three times. The cell-in-cell structures were counted using fluorescence microscopy.

EBV-negative CNE-2 cells pre-stained with 1 μM CellTracker Red dye (Invitrogen) were co-cultured with GFP-Akata cells pre-stained with 2.5 μM CellTracker Green dye (Invitrogen) at 1:10 ratio for 12 h. After washing with pre-cold PBS for three times to remove free GFP-Akata cells, cells were trypsinized to a single-cell suspension and resuspended with PBS containing 10% FBS. Cells were sorted immediately on FACS Aria II (Becton Dickinson, USA), and double-labeled cells with heterotypic cell-in-cell structures were collected by centrifugation for 5 min at 500× g and plated on 96-well plates. Cells were selected in culture medium containing 450 μg/ml G418 for 2 weeks.

CNE-2 or NEPC1-Bmi1 cells were trypsinized to a single-cell suspension and labeled with CellTracker Red dye (Invitrogen). The labeled cells were seeded in 24-well plates with sterile, acid-treated 12-mm coverslips inside at 4 × 10⁴ cells per well in RPMI 1640 medium containing 10% FBS. After 18 h, the culture medium was removed and GFP-Akata or AK31 cells pre-stained with CellTracker Green dye (Invitrogen) at 4 × 10⁵ cells per 0.5 ml culture medium were added into each well. Cells were incubated for the indicated times at 37 °C, 5% CO₂. After co-culturing for different time intervals, non-adherent GFP-Akata or AK31 cells were removed and adherent cells were fixed with 4% paraformaldehyde (PFA). Cell internalization frequency was calculated from a total of 200 cells in at least three independent experiments under a Leica AF7000 fluorescence microscope using a 63× 1.3 numerical aperture PlanApo objective. To confirm whether GFP-Akata cells were completely internalized by CNE-2 or NEPC1-Bmi1 cells, cells were imaged by using a confocal laser scanning microscope (Carl Zeiss Inc) with a 63× objective. Volocity software was used for image acquisition. 4, 6-diamidino-2-phenylindole (DAPI; Invitrogen) was added before the observation to localize the nucleus.

Prior to the start of the experiment, macrophages were labeled with CellTracker Green^™ dye and tumor cells were labelled with CellTracker^™ Red dye, according to manufacturer’s instructions (cat# C7025, C34552, Invitrogen). 1×10⁵ macrophages were cultured with or without 1×10⁵ tumor cells in α-MEM media (supplemented with 0.5% FBS and 300 units/mL of CSF-1) and with either CSF-1R blocking antibody or isotype control antibody at 50 ng/mL (cat# NBP1–43363, clone AFS98, Novus Biologicals; IgG K isotype control cat# 554682, BD Pharmingen). After 24 hours incubation, the experiments were stopped by washing the cells with ice-cold PBS followed by fixing them for twenty minutes in 4% PFA, and permeablizing in 0.1% Triton X-100 in PBS. The cells were stained for VEGF-A using antibodies against mouse VEGF-A (cat# 512809, clone 2G11–2A05, Biolegend) and AlexaFluor647-conjugated secondary antibodies. The intensity of VEGF-A staining in the macrophages was measured by first outlining the macrophages in the green (488 nm) channel in ImageJ/Fiji (69 (link)) and applying that outline to the far-red (647 nm) channel to measure the VEGF-A signal within the macrophage, excluding any VEGF-A signal from the tumor cells.