br antimycotic Thremo The detailed
antimycotic (Thremo). The detailed schedule for the diﬀerentiation of BMDMs is shown in Fig. 2a. Bone marrow cells were extracted from the hind leg and seeded in a culture dish overnight (Day 0). After the floating bone marrow cells were isolated, they were seeded with 20 ng/ ml murine macrophage colony-stimulating factor (M-CSF) at 3.5 × 106 per 100 mm petri dish (Day 1). Then, M-CSF was added daily (Day 2–3) and replaced with fresh media containing 20 ng/ml M-CSF (Day 4 and Day 6). Diﬀerentiation of BMDMs was confirmed by flow cytometry (Accuri C6; BD Biosciences) using anti-F4/80 Mitomycin C (Fig. 2b).
2.3. BMDMs and cancer cell labeling with CellTracker dye
BMDMs and cancer cells were washed with DPBS and re-suspended in culture media at a concentration of 106 cells/ml. CellTracker Green (CMFDA) or CellTracker Deep Red dye was added to 1 ml of cell sus-pension to a final concentration of 1 μM. Note that CFSE (Carboxyfluorescein succinimidyl ester) dye can be used as an alter-native to CellTracker dyes in experimental assays. After incubating for 30 min at room temperature, the cells were washed with PBS and re-
suspended in the culture media.
2.4. Cancer cell labeling with pHrodo-SE
4 T1-Luc, B16F10-Ova, CT26.CL25, and HT29 cancer cells were washed twice with DPBS and re-suspended in DPBS at a concentration of 107 cells per 50 ml. pHrodo-SE dye was added to 50 ml of cell sus-pension to a final concentration of 120 ng/ml. After incubating for 30 min on a rotator (15 rpm) at room temperature, the cells were wa-shed twice with PBS and re-suspended in the culture media.
2.5. Flow cytometry–based phagocytosis assay
For flow cytometry analyses, CellTracker Deep Red–stained BMDMs were plated at a density of 2 × 105 cells per 35 mm petri dish (Falcon). The next day, the stained BMDMs were pretreated with 30 μM Y27632 for 1 h. Then, 4T1-Luc, B16F10-Ova, and CT26.CL25 cells were stained with 1 μM CMFDA and were then co-cultured with syngeneic BMDMs at a ratio of 1:2 in the presence or absence of 30 μM Y27632. After in-cubation for 2 h at 37 °C, the collected cells were pipetted repetitively with PBS to suspend the pellets as single cells and analyzed by flow cytometry (Accuri C6; BD Biosciences) using the FlowJo (v10) software (Fig. 1). The phagocytosis (%) was calculated as the percentage of CMFDA+ cells within Deep Red+ macrophages according to the fol-lowing formula: the number of BMDMs phagocytosing cancer cells (right-upper quadrant, double positive)/total number of BMDMs (right quadrants, green) × 100.
2.6. Fluorescence microscopy–based phagocytosis assay
For microscopic analyses, BMDMs (2 × 105) labeled with CMFDA were cultured in 35-mm glass-bottom confocal dishes (Corning) along with various cancer cells (4 T1-Luc, B16F10-Ova, and CT26.CL25 cells) labeled with pHrodo-SE at a ratio ranging from 1:1 to 1:4 in the pre-sence of phagocytosis agonists (30 μM Y27632 or 10 μg/ml anti-CD47 antibody). Notably, CMFDA-stained BMDMs were pretreated with a ROCK inhibitor, Y27632 (30 μM) for 1 h before phagocytic incubation.
After co-incubation for 2 h at 37 °C, cells were washed several times with basic PBS (pH 10) to remove unengulfed pHrodo-labeled tumor cells. The degree of the phagocytosis of tumor cells was measured by fluorescence microscopy (Nikon) and analyzed by randomly selected six or more microscopic fields per assays (Fig. 1).
Phagocytosis (%) was calculated according to the following for-mula: the number of BMDMs phagocytosing cancer cells/total number of BMDMs × 100. The phagocytosis index (PI) is defined as the following formula based on microscopy images: the number of engulfed cancer cells (pHrodo-SE, red)/total number of BMDMs (green).
The phagocytosis capacity (PC) is defined as the following formula: the number of engulfed cancer cells/the number of BMDMs phagocy-tosing cancer cells.
2.7. Statistical analyses
Data are expressed as means ± standard deviation (SD) for control and experimental samples. The sample size was chosen based on the typical size used in this field. Comparisons between two groups were performed with the Student's t-test using Sigma Plot 10.0 software. P-values < .05 were considered to be statistically significant; individual p-values are indicated in the figure legends.
3.1. Comparison of assay methods for the detection of phagocytosis
To quantify the phagocytosis of cancer cells by macrophages
requires that targeted cells are labeled with a fluorescent dye, such as CellTracker, which emits fluorescence at diﬀerent wavelengths. This phagocytosis assay can be characterized by its sensitivity as well as its simplicity and countability. To determine the phagocytic function of macrophages, fluorescence-labeled target cells were co-incubated for 2 h, and they were dislodged from the plates with harsh pipetting. The extent of phagocytosis was easily quantified by tracing the co-locali-zation of fluorescence using flow cytometry; the percentage of double-stained macrophages (red square) out of the whole macrophage popu-lation (blue square) measures the fraction of the macrophage popula-tion involved in the phagocytosis of target cells (phagocytosis %; Fig. 2c). This phagocytosis assay that utilizes CellTracker dye can also be performed using fluorescent microscopy.