Fluorescence micrographs demonstrating the distribution of inherently fluorescent Janus kinase 3 inhibitors

Published: 21 September 2023| Version 1 | DOI: 10.17632/y7crpxfvcf.1
, Florian Maier,


The dataset contains supplementary images for the publication "Inherent fluorescence demonstrates immunotropic properties for novel Janus kinase 3 inhibitors". In short, cells were incubated with fluorescent JAK3 inhibitors or control fluorophore 13 (appearing in green) as well as stains for lysosomes (appearing in red) or nuclei (appearing in blue). For in vivo samples (tail blood and organs), C57/BL6 mice were treated p.o. with the aforementioned JAK3 inhibitors or the control. Again, nuclei were stained blue and compounds appear in green. File names indicate the test compound used (1-13), the sample type (cell type or organ), incubation time (for in vitro samples), sampling time post treatment (for in vivo tail blood samples) and the level of magnification. "Overlay" indicates a merge of channels for compound fluorescence, lysosome and nucleus stains. "Overview" indicates a merge of singular captures to give an overview of the entire organ sample. HBC = human buffy coat HMC3 = human microglia cells PBMCs = peripheral blood mononucleated cells


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General: Images were acquired using a Keyence BZ-180 fluorescence microscope (Keyence, Tokyo, Japan). For in vitro pictures and closeups of organ tissues, a 100X/a.45 oil-immersion objective lens was used. For each sample within a category, the same exposure and intensity were were used. Haze reduction was applied to the captured images with the corresponding software. In vitro: Cells were pre-incubated for 15 min with Hoechst 33342 solution and Cell Navigator Lysosome Staining Kit (CNLSK), 1:1000 dilution for both, and then incubated with the indicated test substance (c = 10 µM) for 0 min or 120 min. 20 µl of the resulting suspensions were then pipetted onto microscope slides (Roth). Images were acquired with a Keyence BZ-810 fluorescence microscope (Keyence, Tokyo, Japan) using the same exposure and intensity for each sample within a given category. Nuclei (stained by Hoechst) were captured in the DAPI Filter (excitation wavelength 360/40 nm, detection wavelength 460/50 nm) and appear blue. Lysosomes (stained by CNLSK) were captured using the TexasRed filter (exc. wavelength 560/40 nm, det. wavelength 630/75). The fluorescent test compounds were captured using the GFP filter (exc. wavelength 470/40 nm, det. wavelength 525/50 nm). Images were then processed with the corresponding software. In vivo: 7-8 week old C57BL/6 male mice were treated orally with vehicle (0.5 % citric acid, 5 ml/kg) or 24 µmol/kg of the test compounds. Blood was taken from the tail vein at predetermined time points into anticoagulant-containing tubes and centrifuged at 8000 rpm for 8 min to obtain tail plasma. 2-4 h post treatment (4 h for compound 13, 2 h for others), animals were sacrificed and organs were taken for subsequent analysis.


Eberhard Karls Universitat Tubingen


Lysosomes, Fluorescence Microscopy, Compound Uptake, Tyrosine Kinase Inhibitor