Fx630

The schematic design and procedure of the on-chip free-flow method are shown in Figure 1B,C. First, $5.0\times {10}^4$ cells were placed in the 35 $\mathrm{m}$ $\mathrm{m}$ cell-culture dish (AGC TECHNO GLASS Co., Ltd., Shizuoka, Japan) with 2 $\mathrm{m}$ $\mathrm{L}$ DMEM and incubated for 3 h at 37 °C under 5% CO₂ to allow them to adhere to the bottom of the dish. Then, $2.0\times {10}^6$ antigen beads were added to the dish carefully, and it was placed on the inverted microscope (IX71; 10× obj. lens; Olympus, Tokyo, Japan) stage with a slight tilt (see Figure 1C) to form $0.55$   $\mathsf{\mu }$ m/s of antigen flow before phagocytosis of the macrophages had begun. Since macrophages adhered to the bottom of the dish, tilting the cultivation dish generated a flow of antigen beads to the macrophages.
The observation was started as soon as the dish was set on the stage, and the concentration of antigen beads and the percentage of phagocytic cells in the observed area at each time were measured. During the experiment, the dish was placed in an appropriate environment for cell activity (37 °C, 5% CO₂) by a stage-top incubator (Tokai Hit., Co, Ltd., Shizuoka, Japan). The microscope was equipped with a charge-coupled device camera (FX630, CCD camera; Olympus, Tokyo, Japan) to take micrographs at each time.

The spermatozoa placed on a glass bottom dish were analyzed at 3,700× using an inverted microscope equipped with Nomarski differential interference contrast optics (IX71, Olympus, Tokyo) and a video system (FX630, Olympus, Tokyo). A 60-× (1.42 numerical aperture) objective lens was used with oil. Images of the spermatozoa were captured and stored on a video system using an image-filing software program, FlvFs (Flovel, Tokyo). We spent 30 to 60 minutes capturing and analyzing the images of each ejaculate. The spermatozoa were classified into three groups: (I) those bound to MH61-beads after the acrosome reaction at 24 hours of incubation regardless of the motility, (II) motile spermatozoa that did not bind to MH61-beads, and (III) immotile spermatozoa that did not bind to MH61-beads. At least 500 spermatozoa per ejaculate and 100 spermatozoa per each group were evaluated using the high-magnification microscope [14 (link)]. A nuclear vacuole was defined as “large” if the maximum diameter of the vacuole was more than 50% of the width of the sperm head [14 (link)]. Using this system, we evaluated large nuclear vacuoles (LNVs) not only in motile spermatozoa but also in immotile spermatozoa (Figure 1).
The percentage of spermatozoa with LNVs was calculated for each sample and compared between groups I, II, and III.