Cellvue nir780

Spheroids of LEC were generated from 2000 cells in hanging drops in ECGM2 containing 20% of methylcellulose (M6385, Sigma Aldrich). As previously described in ref. ⁵⁶ , spheroids were embedded in a collagen gel (08-115, Merck, Germany) and cultured in the respective medium of LEC. Depending on experimental settings, cells were stimulated with 100 ng/mL VEGF-C, treated with 25 µM CQ, 20 mM ammonium chloride (NH4Cl), and 100 µM leupeptin or supplemented with 20 mM AC and cultured for 48 h to analyze sprouting. For the mixed spheroids, LEC were collected 48 h after transfection and labeled with cell membrane labeling lipophilic dyes CellVue™ Jade and CellVue™ NIR780 (88-0876-16 and 88-0875-16 respectively, Thermo Fisher Scientific). Spheroids containing equal amounts of si CTRL transfected cells (green) and si ATG5 transfected cells were generated, embedded, and analyzed after 48 h. Images were taken in an inverted microscope (IX83, Olympus) and an analysis for number of sprouts per spheroid was performed using NIH ImageJ software.

Following neutrophil isolation, cells were washed in serum free media. Cells were counted and resuspended at concentration of 2 × 10⁷ cells/mL in Diluent C (supplied in the CellVue labelling kit, Thermo Fisher Scientific). For a final labelling concentration of 2 μM of dye, a working solution was prepared by adding 4μL of the 1 mM dye stock to 1 mL of Diluent C. Cells and dye solution were mixed together followed by a 3-min incubation time at room temperature. Cell labelling was stopped by adding an equal volume of 100% fetal bovine serum (FBS) and incubated for 1 min at room temperature. Cells were then washed by centrifugation and the supernatant was discarded. The cell pellet was resuspended in complete media and then washed a further 3 times in PBS to remove residual Diluent C and any unbound dye prior to intravenous administration.
The above protocol was applied for both CellVue NIR815 (Thermo Fisher Scientific catalogue number 88-0874-16) and CellVue Burgundy (Thermo Fisher Scientific, catalogue number 88-0872-16). However, for the CellVue NIR780 (Thermo Fisher Scientific, catalogue number 88-0875-16) a final concentration of 4 μM was used to achieve the same IVIS signal to that of CellVue Burgundy.