5 μl syringe

The scAAV5-smCBA-hRPE65 vector as used in previous studies was used to deliver RPE65 gene in rd12 mice, with the same method of subretinal injections at age P14 [18 (link), 21 (link), 22 (link)]. Animals were prepared with pupil dilation and general anesthesia. A 30.5-gauge disposable needle was used to make a small incision in the cornea within the pupil area. Then a 33-gauge, unbeveled, blunt needle mounted on a 5 μL syringe (Hamilton Co., Reno, NV) was introduced through the corneal incision to reach the subretinal space in the inferior central region, avoiding touching the lens and penetrating the neuroretina. One microliter of vector suspension (1 × 10¹³ genome containing particles/mL) containing 1% fluorescein was injected slowly in the subretinal space in the right eye of rd12 mice. The injected retinal area was visualized by fluorescein positive subretinal blebs demarking the retinal detachment and more than 95% retinal detachment indicates successful injection. After injection, 1% atropine eye drops and 0.3% tobramycin-dexamethasone eye ointment (Alcon Laboratories Inc., Fort Worth, TX) were given 3 times a day for 3 days. Animals with any complications, including iris-cornea adhesion, iris or retinal hemorrhage, and lens injury, were excluded from the experiment.

A home-made polydimethylsiloxane flow cell was made using the same method reported elsewhere19 21 . Briefly, glass microscope slides were cleaned and submerged in a solution of toluene (50 mL) and OTS (15 μl) for 1 h at 50 °C. The glass slides were subsequently washed with toluene, acetone, ethanol, and deionized water (DIW) and dried with a stream of nitrogen, and then wrapped in aluminum foil and stored at 40 °C under vacuum for 24 h. The OTS induces the perpendicular alignment of the LC. A copper TEM grid was placed on the surface of the 12 × 8 mm² OTS-coated glass that was glued to another common slide glass with epoxy. A 1 μL drop of E7 was placed on a TEM grid using a 5 μL syringe (Hamilton Co., Reno, Nevada, USA). Excess E7 was removed with a capillary tube to obtain a uniform thin film. The two slide glasses spaced with silicon rubber (2 mm) were then clipped with binder-clips. E7 rather than 4-cyano-4′-pentylbiphenyl (5CB) was used owing to the need to facilitate a high nematic-to-isotropic transition. The inlet and outlet ports for exchanging the solutions were made with needles that were punched through the silicon rubber. The internal volume of the flow cell was 0.4 mL.

For BM sinusoid cell labeling, WT or PLZF-GFP mice were injected intravenously with anti-CD45.2⁻ AmCyan antibody (1 μg; clone 104, BioLegend). Mice were euthanized 2 min after the injection, and BM cells were stained for flow cytometry to determine the numbers of indicated ILC progenitor cells. For BM cell labeling with CellTracker Deep Red (Thermo Fisher Scientific) (33 (link)), the skin above the skull or tibia was cut open, and small holes were made with a 30-gauge needle. Then, 3 μl of diluted CellTracker was slowly injected into each site by using a 5-μl syringe (#65 Hamilton Co., Reno, NV, USA) and a custom 34-gauge blunt needle (RN 0.375″ PT3, Hamilton). The skin incision was sutured, and mice were euthanized for flow cytometry analysis 24 to 48 hours after the microinjection. Emigration rate (%) for each organ was based on the ratio: [Frequency of CellTracker-labeled cells of CD45⁺ cells in blood or colon]/[Frequency of CellTracker-labeled cells of CD45⁺ cells in the BM].