30g insulin syringe

Lymphatic drainage was measured as described previously [39 (link),40 (link)]. In brief, ACKR3^iΔLEC and ACKR3^WT animals were placed in an IVIS imaging system (Caliper Life Sciences) and 0.05 nmol/g IRdye800-PEG-20 were injected s.c. using a 30G insulin syringe (Terumo, Tokyo, Japan) with the ears taped down flat at the rim of the ear. The fluorescence intensity in the ear skin (λex = 745 nm, λem = 800 nm, exposure time 4 seconds, binning 2) was measured at 0, 1, 2, 4, 6 and 24h. For analysis, ROIs were drawn around the ears and the average fluorescence intensity was measured in each ROI using Living image 4.0 software (Caliper Life Sciences, Hopkington, USA). After subtraction of the fluorescence intensity of uninjected ears, used as blank measurement, the average fluorescence intensity in each ROI was normalized to the initial average fluorescence intensity at time 0. The normalized average fluorescence intensities were plotted against time in Excel. Data points were fit according to first- order kinetics and the corresponding half-life (t_1/2) was determined (T_1/2 = ln_2/k). Lymphatic drainage in TPA-induced acute inflammation was measured one day after TPA-application and after determination of an increase in ear thickness.

Lymphatic vessel drainage function was assessed by measuring the clearance over time after intradermal skin injection of the infrared probe P20D800 (Karaman et al, 2015 (link)). Mice were anesthetized with isoflurane (2%), and 3 μl of 3 μM P20D800 was injected intradermally in the ears with a 30G insulin syringe (Terumo). For mice with D7 tumors, injection was performed in the peritumoral area. The mice were then positioned in a whole-animal fluorescence imaging system (NightOWL II; Berthold Technologies), and images were acquired with the following imaging settings: λ_ex: 745 nm, λ_em: 800 nm, and an exposure time of 4 s. Subsequent images were acquired of the ears at 1, 2, 3, 4, 6, and 24 h after injection. Mice were allowed to wake up and move freely between imaging time points. Fluorescence signal intensities were adjusted to baseline ear signals before injection of tracers to calculate tissue enhancement values. The fluorescence intensity values over time were fit to a one-phase exponential decay model in GraphPad Prism 7.0 software with lymphatic clearance expressed as decay constant k (expressed in h⁻¹) or as half-life (expressed in h) using the following equations: ${Normalized\hspace{0.17em}Fluorescence\hspace{0.17em}Intensity=e}^{-kt}$ $HalfLife=In\left(2\right)/k$

Mice were anesthetized with isoflurane (2%), and 5 μl of 50 pg/μl VEGFA or 5 μl of 5 ng/μl VEGFC were injected into the left ear dermis using a 30G insulin syringe (Terumo). An equal volume of PBS was injected into the right ear as a control. The injection site was visually marked using a marker pen. Ears were collected after 15 and 30 min, followed by fixation in 4% PFA and whole-mount immunostaining.