Diff quik stain set

For invasion assays, cells were treated with the indicated siRNAs. After 48 h transfection, cells were trypsinized, counted with a Coulter counter and diluted to a desired concentration (H1299: 0.5 × 10⁵; H838: 1 × 10⁵. 0.5 ml cell suspension per well). Cells were seeded onto basement membrane matrix Boyden chambers (8-mm pore size, BD) present in the insert of a 24-well culture plate (Matrigel was purchased from BD Company). 20% FBS was added to the lower chamber as a chemoattractant. After 48 h, the non-invading cells and EC matrix were gently removed with a cotton swab. Invasive cells located on the lower side of the chamber were stained with Diff-QuikTM Stain Set (SIEMENS), air dried and photographed.

For invasion assays, cells were treated with the indicated siRNAs. After 48 h transfection, the cells (H1299: 0.5×10⁵; H838: 1×10⁵) were collected and resuspended in serum-free medium. These cells were seeded onto basement membrane matrix Boyden chambers (8-μm pore size, BD) present in the insert of a 24-well culture plate. The lower compartment of Boyden chambers was filled with 600 μl medium containing 20% FBS as chemoattractant. After 48h incubation, matrigel and non-migrating cells were removed from the top chamber with cotton swabs. Invading cells on the bottom were stained with Diff-QuikTM Stain Set (SIEMENS). After air drying, the number of invaded cells per chamber was counted in five separate fields under a microscope and the average values calculated.

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.

Migration assays were performed using Costar 24-well Transwell plates (Corning, Tewskbury, MA). AVMECs (passages 6–10) and HBMVECs (passages 9–10) were plated at 100,000 cells/well in 100 μL of EGM2 media (Lonza, Walkersville, MD) with 0.1% serum in the upper chamber. In the lower chamber, either EGM2 with 0.1% serum (negative control), EGM2 full serum media (positive control), or EGM2 with 0.1% serum supplemented with EphrinB2-FC recombinant 250 ng/mL (R&D Cat. No. 7397-EB-050, Waltham, MA) or 100 nM EphB4 inhibitor^{19 (link)} (Sigma, NVP BHG 712, St. Louis, MO) were added to the lower chamber to act as a chemoattractant. After 24 h, cells remaining in the upper chamber were aspirated; cells in the bottom chamber were fixed, permeabilized, and stained using Diff-Quik Stain Set (Siemens, Malvern, PA) as previously published^{20 (link),21 (link)}. Invasion assays were performed in a similar fashion as above using Matrigel invasion chambers (Thermo Fisher Scientific, Waltham, MA). Experiments were performed in duplicate for each cell line, with a total of ten high-power fields analyzed per cell line. Representative images were captured at ×100, and the number of migrated (or invaded) cells was quantified using ImageJ software (National Institutes of Health, Bethesda, MD).

Strains of B. divergens were isolated from bovine blood during the acute phases of babesioses as described earlier [25 (link)]. 11 isolates of B. divergens from different geographical locations within France were cultivated and cloned by limited dilution [26 (link)]. The first two digits in the description of each clone (Additional file 1: Table S1) refer to the French county of origin. Isolate Rouen 87 originated from human blood [27 (link)]. Babesia divergens isolates were cultivated in vitro in a suspension of bovine erythrocytes obtained from a parasite-free cow (serologically negative and culture tested) as described [25 (link), 26 (link)]. Parasitemia was monitored using the commercial Diff-Quik Stain Set (Siemens) and RBC smears.