Cascade blue

To create a chmp7 mutant zebrafish line, a guide RNA targeting chmp7 (ENSDARG00000041362) exon 2 was generated according to Gagnon et al.¹⁷ . One-cell stage embryos were injected with a mixture containing: 150 ng/µL guide RNA, 5 µg/µL Cas9 protein (PNA Bio, Newbury Park, CA, USA), 20 µM STOP cassette, 0.25 µL Phenol Red, 0.25 µL Cascade Blue (Molecular Probes, Waltham, MA, USA), and ultra-pure H₂O to a final volume of 2.5 µL. Embryos were selected for Cascade Blue, indicating successful injection, at 24 hpf and raised to adulthood. F₀ founders were identified by outcrossing to TU fish, collecting DNA from 15 to 20 offspring, and the pooled DNA used as a template for PCR amplification of the region surrounding the target site. Polyacrylamide gel electrophoresis was used to visualise heterodimers. Founders were outcrossed to wildtype fish, and F₁ individuals screened for the presence of mutations using PCR and gel electrophoresis. The mutation was determined using Sanger sequencing. Experiments utilised F₃ and subsequent generations. Guide RNAs and primer sequences are presented in Supplementary Table 2. Genotyping was performed using allele-specific KASP fluorescence assays (LGC Biosearch Technologies, Teddington, Middlesex, UK).

To measure apparent paracellular permeability (P_app), 50 g/mL Cascade Blue (3 kDa, Thermo Fisher Scientific) was added to the antibiotic-free expansion media in the top epithelial channel with a flow rate of 30 μL/hr. Every 24 hours, the effluents from the epithelial and endothelial channels were collected. The concentration of Cascade Blue that diffused across the membrane into the endothelial channel effluent was quantified with a Varioskan LUX multimode microplate reader (Thermo Fisher Scientific) with excitation at 400 nm and emission at 425 nm. The following formula was used to calculate P_app (cm/s) as in Kasendra et al. (16 (link)): P_app = (Endothelial_Output × Flow Rate)/(Epithelial_Input × Surface Area), where Endothelial_Output is the concentration (mg/mL) of Cascade Blue from the endothelial channel effluent, Flow Rate is the fluid flow rate (mL/s) of the media through both channels, Epithelial_Input is the concentration (mg/mL) of Cascade Blue originally added to the epithelial channel media, and Surface Area is the area (cm²) of the channel where coculture occurs.