Diaphot 200 microscope

The in vitro scratch assay was performed on LNCaP cells stably transfected with the control pcDNA3.1⁽⁺⁾ vector or vector containing GPD2 (untagged form) according to [34 (link)]. Cells were seeded at the density of 3 × 10⁶ cells/well in 6-well plate and maintained in the cell culture medium. The plates were kept in 5% CO₂ atmosphere at 37 °C for 24 h to form a monolayer. A scratch was created by scraping a straight line using a 10 µL pipet tip. Cells were once washed with 1 mL of culture medium to remove debris and images were acquired at 0 and 24 h using a Nikon Diaphot 200 microscope, objective 10× (Nikon, Tokyo, Japan). To obtain the same field during image acquisition, the markings were created close to the scratch using an ultrafine marker.
The wound area was measured using ImageJ software following the protocol described by [35 (link)]. The rate of cell migration was calculated based on the change in % area covered with cells between time 0 and 24 h.

Shortly before each experiment, cells (i.e., GH₃ or INS-1 cells) were dissociated and a few drops of cell suspension was transferred to a home-made chamber mounted on the fixed stage of an inverted Diaphot-200 microscope (Nikon, Tokyo, Japan). They were immersed at room temperature (20–25 °C) in normal Tyrode’s solution, the composition of which is described above. We fabricated the recording electrode from Kimax-51 glass capillaries (#34500; Kimble, Vineland, NJ) using a PP-830 vertical puller (Narishige, Tokyo, Japan) in which a two-step pull mechanism was applied, and their tips were fire-polished with a microforge (MF-83, Narishige). During the measurements, the electrode with tip resistance ranging from 3 to 5 MΩ, which was firmly inserted into holder, was maneuvered by use of a WR-98 micromanipulator (Narishige). Patch-clamp experiments operated under voltage- or clamp-clamp mode were carried out by using an RK-400 patch-clamp amplifier (Bio-Logic, Claix, France) connected with a personal computer [43 (link)]. Shortly before giga-seal formation was achieved, the potentials were commonly corrected for the liquid junction potential that generally developed at the pipette tip, as the composition of internal solution was different from that in the bath.

Fixed spleen tissues were also used for brightfield imaging. Large slices (~2 mm x 3 mm by x 0.5 mm) of tissue were cut with a microsurgical scalpel and placed on glass slides. Specimens were imaged with a Diaphot-200 microscope (Nikon, Inc.) using a 4x objective lens in brightfield mode. Images were recorded digitally.

HeLa S3 cells adapted to adherent culture were maintained in DMEM (4.5 g/L glucose) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, and 0.37% sodium bicarbonate at 37 °C in 5% CO₂. HeLa Tet-Off Advanced cells (Clontech) were maintained in DMEM supplemented with 10% Tetracycline-free FBS (Clontech), 2 mM L-glutamine, 0.37% sodium bicarbonate, and 100 μg/mL G418 to maintain expression of the transactivator.
Induced Pluripotent Stem (iPS) cells (System Biosciences Cat # SC101A-iPSC, Lot # 110415-01) were cultured on Matrigel (BD Biosciences) in mTesR1 media (STEMCELL Technologies) at 37 °C in 5% CO2. Human foreskin fibroblasts (HFFs; System Biosciences Cat # SC101A-HFF, Lot # 110509) were maintained in DMEM (4.5 g/L glucose) supplemented with 2 mM L-glutamine, 0.1 mM non-essential amino acids, and 10% FBS at 37 °C, 5% CO₂.
HeLa Tet-Off Advanced cells were transfected with reporter plasmids using jetPRIME (Polyplus) prior to allowing them to adhere to the dish. A ratio of 3 µL reagent to 1 µg of plasmid was employed according to the manufacturer’s recommendations. Cells were harvested 24–48 hr after transfection. Transfections were evaluated for ZsGreen expression using a Nikon Diaphot 200 microscope with a GFP-B filter cube (Nikon). Transfection efficiency was generally >70% at ~48 hours post transfection.