Rhodamine dextran solution

The KV fluid dilution assay was performed by aspirating the vesicular fluid into a micropipette previously filled with Danieau’s solution 1 x (58 mM NaCl, 0.7 mM KCl, 0.4 mM MgSO4, 0.6 mM Ca(NO3)2, 5.0 mM HEPES pH 7.6) diluted in 10,000 MW rhodamine-dextran solution (1:4; Sigma-Aldrich). The mixed solution was injected again into the KV and only embryos with rhodamine-positive KV’s were selected for the experiment. For viscosity manipulation, the micropipette was filled with Danieau’s solution 1 x containing 1.5% (w/v) of methylcellulose (M0555, Sigma). Aspirated KV fluid and methylcellulose were then injected again into the KV lumen until reaching the KV volume observed before the manipulation.

Before two-photon microscopy, 1 × 10⁶ CD8⁺ T cells from dCLNs of mice injected with parental or LM cells were isolated, labeled by 0.5 μM CFSE (Thermo Fisher, Cat# C34554) for 15 min at 37℃, and transferred to recipient mice via caudal veins. 24 hr after T cell transfusion, mice were anesthetized, and thinned skull windows were prepared. Mice were injected with 100 μL rhodamine dextran solution (100 mg/mL, Sigma, R9379) where indicated to visualize blood vessels. Images were captured in z-stacks of 10–30 planes (1 μm step size) using an Olympus FVMPE-RS two-photon microscope.