Ix71 inverted microscope

Colony formation, wound healing, and transwell assays were conducted as in prior reports [14 (link)]. To measure colony formation, we added 200 GC cells per well to a 6‐well plate, followed by culture for 14 days. Cells were fixed with 4% paraformaldehyde, stained with 1% crystal violet, and colonies comprising > 50 cells were counted. For wound healing assays, cells were seeded on 6-well plates, grown to confluency, and scratched with a 200 μL pipette tip. Wound recovery was observed under an IX71 inverted microscope (Olympus) after 0 and 48 h. In transwell assays, cells were seeded on 24-well transwell plates (Corning, New York, NY) to measure their invasive capacity. Inserts were coated with 60 µl of Matrigel (BD Biosciences, Franklin Lakes, NJ). After 24 h, invaded cells were fixed and stained with 0.1% crystal violet solution containing formaldehyde. The numbers of invaded cells were counted in five randomly-selected fields under an IX71 inverted microscope (Olympus).

To further characterize the effects of the four SP-D variants on the agglutination of MH-S cells after mycobacterial infection as described above, micrographs were taken 2 days after infection using an Olympus IX-71 inverted microscope. The area of the agglutinates was measured using ImageJ 1.39g software (National Institutes of Health, Bethesda, MD, USA). The diameter was estimated from the area based on the circular form of the aggregates according to the formula Area = (R × D²)/4, where R = ratio of a circle’s circumference to its diameter and D = diameter. To determine the effects of the four variants of SP-D on macrophage cell migration, 2.5 × 10⁴ MH-S cells were seeded in a 24-well cell culture plate and infected with M. bovis BCG as described above. The culture medium was harvested 2 days after infection and added into a new 24-well cell culture plate equipped with Millicell Hanging Cell Culture Inserts (3 µm pore size, Millipore). Each insert was seeded with 5 × 10⁴ MH-S cells. After 1 day of culture, the inserts were removed and observed using an Olympus IX-71 inverted microscope to count the cells migrated through the membrane. The chemoattractive MH-S cells were quantified using ImageJ 1.39g software.

Ten microliters of anticoagulant blood were smeared on each glass microscope slide. Then, the slides of blood cells smears were dried and fixed in methanol for 5 min at RT, and then immersed into May-Grünwald solution for 3 min, then immersed into PBS solution (pH 6.8) for 1 min. Finally, the slides were stained with Giemsa solution (diluted 20 times with the PBS) for 10 min and washed with ddH₂O for 30 s, air-dried and visualized under Olympus IX71 inverted microscope (Tokyo, Japan).
After the NOD/SCID mice were sacrificed, the spleen was harvested and fixed with 4% paraformaldehyde. Then these tissues were dehydrated in a series of alcohol, embedded in paraffin and sliced into 5-μm sections. Hematoxylin and eosin staining was carried out according to the routine staining method. Briefly, the sections were dewaxed, rehydrated in alcohol, stained with hematoxylin for 15 min, differentiated, and then stained with eosin for 3 min, dehydrated in alcohol and xylene, and mounted. The sections were examined under Olympus IX71 inverted microscope (Tokyo, Japan).

Primary HTM cells (ScienCell Research Laboratories, Inc., Carlsbad, CA, USA; see http://www.sciencellonline.com for details) were cultured in TM Cell Medium (TMCM; ScienCell Research Laboratories) consisting of basal medium, 2% fetal bovine serum, 1% TM cell growth supplement (TMCGS) and 1% penicillin/streptomycin solution, at 37°C in an atmosphere of 5% CO₂.^{7 (link),8 (link)} The medium was changed every other day until the cells were approximately 90% confluent. Subsequently, cells were passed sequentially in a 1:4 ratio and maintained in the same medium. Cells of passages 4 to 5 were used in the experiments.
In a 24-well plate, HTM cells (5 × 10³ cells) were transduced with LV-C3-GFP or LV-GFP at multiplicities of infection (MOIs) of 80, 160, 400, 640, 800, and 1130. MOI was determined by simply dividing the number of viral particles added (mL added × TU/mL) by the number of cells added per well. The optimal MOIs were evaluated through detecting the expression of GFP and changes in morphology by using an IX71-Olympus inverted microscope with Nomarski optics (IX71; Olympus, Tokyo, Japan).

NOZ and OCUG-1 cells were seeded in 6-well plates (5 × 10⁵ cells per well) to form a confluent monolayer. After culturing for 24 h, a sterile 1,000 μL pipette tip was used to scratch the monolayer in all wells. The cells in each well were then treated as described. The distance between cells was taken at the same position at 0 and 24 h using an IX71-Olympus inverted microscope (Olympus, Tokyo, Japan) at 10 × magnification. The wound healing index is expressed as a percentage of the wound area at 24 h divided by that at 0 h.