G1020

According to previous study^{66 (link)}, 32Dcl3 cells with a concentration of 2 × 10⁵ cells /ml were collected, washed twice with PBS to remove IL-3, and then resuspended in the induction medium containing complete RPMI 1640 medium supplemented with 25 ng/ml of G-CSF (#78012_C, Stem Cell). Under the condition of the above, 32Dcl3 cells were harvested at sixth day. On the sixth day, cells were collected, and morphological evaluation was carried out using Wright-Giemsa staining solution (G1020, Solarbio) according to the manufacturer’s protocol. The morphology of cells was observed by optical microscope (Nikon Eclipse Ni; Japan).

The caudal epididymis was removed from the anesthetic adult mice treated with or without LY411575 for 35 days and cut by eye scissors into small bulks. Next, these tissues were submerged in 500 μl of Tyrode’s salt solution (T2397, Sigma, U.S.A.) at 5% CO₂, 37°C incubator for 15 min. The sperms swam out into the salt solution after incubation, and were obtained by centrifugation at 600 g for 5 min at room temperature. Next, the sediments were re-suspended with 1 ml of PBS. Two aliquots of suspension (each 10 μl) were taken out to perform with computer-assisted sperm analysis software (CASA software) as previously described and Giemsa staining (G1020, Solarbio, China) was performed according to the production manual [16 (link)]. The length of the sperm midpiece was measured using ImageJ software.

Mice were euthanized by CO₂. Then, the lungs were washed with cold PBS. Afterwards, the BALF samples were centrifuged and then suspended in PBS, and the total number of cells was counted. Later cell medium was centrifuged, and the inflammatory cell counts were analyzed with Wright–Giemsa staining (^#G1020, Solarbio).

The slides of bone marrow (BM) and peripheral blood (PB) from mice were stained with Wright’s Giemsa stain solution (Solarbio, G1020) according to the manufacturer’s instructions and were then observed or photographed under the microscope (Nikon, Japan).

Cell morphology was assessed using slides prepared with a cytocentrifuge (Cytospin 4, Thermo Scientific) at 400 rpm for 5 min, followed by Wright–Giemsa staining (G1020, Solarbio Science & Technology). The brightfield slides were scanned with NanoZoomer S360 (Hamamastu Photonics) at 400x. The images were recorded and analyzed using NDP.view 2.9.22 RUO. Briefly, the diameter of 50 randomly selected cells from each group were averaged and then compared between the groups.

The precipitated cells in BALF were yielded through the centrifugation at 400 × g for the measurement of the numbers of total cells and neutrophils in BALF. Precipitated cells were resuspended in PBS and sowed on 15‐mm glass slides. After cells were maintained for 1 h at 37°C to stick to the slides, Wright's‐Giemsa staining (catalogue number: G1020, Solarbio) was performed to count the numbers of total cells and neutrophils.

The peripheral blood white blood cell count method was performed as described previously (18 (link)). Briefly, fresh blood was collected from the wing vein and 10 µl of which smeared on microscopic glass slides for each bird. Blood smears were air-dried and then stained with Wright’s Giemsa solution (G1020, Solarbio, Beijing) according to the manufacturer’s instructions. According to schematic diagram, two hundred leukocytes (heterophils, lymphocytes, and monocytes) were counted using a Leica DM500 microscope with a magnification of 1000× immersion oil. The percentage of monocytes was equal to the number of macrophages to the total number of leukocytes (heterophils, lymphocytes, and monocytes). H/L was the ratio of heterophils to lymphocytes.