Diff quik stain set

Cell concentrations from the BALF were determined via hemocytometer. A Cytospin 4 (Thermo-Fisher Scientific) was used to deposit cells from the BALF onto glass slides. Cells were then fixed and stained using a Diff-Quik Stain Set (Siemens). Relative percentages of macrophages, neutrophils, eosinophils, or lymphocytes per 500 cells were then identified using a light microscope. The percent of macrophages visually containing U-MWCNTs or Z-MWCNTs per 100 cells per mouse was also determined.

During dissection, blood was collected by cardiac puncture using a 1 ml heparinized syringe. To determine differential white blood cell count (WBC), a blood smear was made on a glass slide by placing a drop of blood on one slide surface and using a second glass slide to push the blood forward from the edge of the drop, and left to dry for 24h. SIEMENS, Diff-Quik™ Stain Set was used for rapid differential staining. Slides were dipped 5 times in a methanolic fixative to stabilize cellular components, then dipped 10 times in a buffered solution of eosin Y and 7 times in a buffered solution of thiazine dye (methylene blue and azure A). Finally, slides were rinsed in distilled water, dried and stored at room temperature. WBCs were identified as monocytes, neutrophils, basophils, eosinophils and lymphocytes. A total of 100 cells were randomly counted in each slide and the data are presented as % of the presence of each cell type.

The viability of LV control and miR-320c HCT116 cells was determined using alamarBlue assay as previously described [10 (link)]. All assays were carried out with appropriate controls. Briefly, 5000 cells were cultured in a 96-well plate and cell viability was measured at the indicated time points by adding 10% volume alamarBlue assay reagent and measuring absorbance at 570λ. The colony forming ability of HCT116 cells transduced with miR-320c was determined using clonogenic assay as previously described [35 , 36 (link)]. Briefly, LV control or miR-320c HCT116 cells were seeded in 12-well plates in different serial dilution (1:2 to 1:64). Initial seeding density was 0.015 × 10⁶ cells per well, and incubated at 37°C under 5% CO2 for 10 days. The plates were then washed and stained with Diff-Quik stain set (Siemens), and the plates were scanned and number of colonies was observed under microscope. The fraction of surviving cells was estimated by comparison of miR-320c to LV control cells. The experiment was done twice in duplicate. Furthermore, the clonogenic assay was conducted to examine the effect of 5-Fluorouracil on colony formation in both cells. A total of 1 × 10⁶ cells were seeded in T25 flask. After 48 hours of exposure to 1.5 μM of 5-Fluorouracil, the cells were trypsinized and reseeded in 12-well plates as described above to observe the effect of the drug.