Wright giemsa

According to our previous study (35 (link)), the bronchoalveolar lavage fuid (BALF) samples were centrifuged at 800×g for 5 minutes. The supernatant was collected and the protein content was determined using the BCA protein assay kit (Thermo Scientific, Rockford, Ill). The cell pellet was resuspended in 100μL PBS and then cells were stained with Wright-Giemsa (Solarbio, Beijing, China) according to the manufacturer’s protocols. The number of inflammatory cells were quantified using a light microscope. Under the microscope, 200 cells/slice were counted at ×40 magnification.

Twenty‐four hours following the final OVA challenge, the mice were sacrificed. PBS (100 mL) was used to wash the pulmonary circulation. To collect the BALF, right bronchus was ligated, and left bronchus was flushed three times with 3 mL of PBS via a catheter; the fluid recovery rate was 80%. Cell suspension was centrifuged at 500 g for 10 min at 4°C. Cell pellet was resuspended in 1 mL of normal saline. A total number of cells in 0.05 mL BALF were counted with a hemocytometer (Baxter Diagnostics, USA). The inflammatory cells were dried and stained with Wright-Giemsa (Solarbio, Beijing, China) following the manufacturer’s instructions and were counted based on conventional morphological criteria. Differential cell counts were obtained by counting at least 400 cells per slide. All counts were conducted with blind methods. The remaining BALF supernatants were maintained at −80°C for next use.

Antibodies against PIM1, p-Smad2 (S467), p-Smad3 (S423 and S425) and p-c-Myc (S62) were purchased from Abcam (Cambridge, MA, USA). Antibodies against E-cadherin, N-cadherin, Vimentin, ZEB1, ZEB2, Snail1, Snail2 (Slug), Twist, MMP2, MMP9, Smad2, Smad3, c-Myc, PCNA, Ki67 and GAPDH were purchased from Proteintech Group (Chicago, USA). Wright-Giemsa was purchased from Solarbio (Beijing, China). The inhibitors 10058-F4 and SGI-1776 were purchased from MedChem Express (New Jersey, USA). TGF-β was purchased from PeproTech (New Jersey, USA). The Alexa Fluor 488- and 594-conjugated secondary antibodies were purchased from Invitrogen (CA, USA).

The lungs were lavaged via a cannula inserted into the trachea. To gain the fluid with cells, 2 × 1 mL aliquots of saline were instilled and then centrifuged at 2000 rpm for 10 min at 4 °C. The supernatants were obtained for ELISA analysis. Then, resuspended cells were counted in a hemocytometer. Cells were coated on glass slides and stained with Wright–Giemsa (Solarbio, Beijing, China) solution. Cells were differentiated by morphological criteria.

The mice were sacrificed within 24 h of the last OVA challenge, followed by a tracheal cannula. For BALF collection, the trachea and right lungs were lavaged three times with 0.3 ml ice-cold PBS. The BALF was centrifuged at 1000 rpm for 10 min at 4°C, and the supernatant was stored at –80°C for further cytokine analysis. The sedimented cells were suspended in PBS for total inflammatory cell count and stained with Wright-Giemsa (Solarbio, Beijing, China) for differential cell counts. A total of 200 cells were counted on each slide.

Macrophages were seeded in the 6-well plate first, after the macrophage medium was removed, 1.0 × 10⁵ CNE2/5–8F cells were resuspended well with 2 ml RPMI 1640 complete culture, after 2 h, removed and washed with PBS to remove unphagocytosed CNE2/5-8F cells, followed by adding 1 ml of Wright-Giemsa (Solarbio Science & Technology, G1020) solution to each well for 5 min.