Rhodamine b dextran

Mean blood flow velocity was measured by the analysis of video footage from micro-endoscopic measurements of the wild-type mouse cheek. For the measurement of mean blood flow velocity, rhodamine-B dextran (70 kDa, Sigma) was injected and used to visualize the blood vessels. In post-processing, erythrocytes were identified and tracked manually over the course of 0.2 s of video. For each wild-type mouse, red blood cells were tracked from different positions, resulting in a total of 5 measurements during each measurement day. Vascular flow data was presented as mean distance ± standard error in the mean over 5 measurements.

After varying treatments as specified in the main text, confluent AC16 human cardiomyocytes seeded on the coverslips were washed three times with 37 °C PBS. They were then plated into 35-mm dishes containing DMEM F-12 cell culture medium (Invitrogen Gibco, Carlsbad, CA, USA). Subsequently, 2 ml PBS containing the low molecular weight gap junction-permeable fluorescent dye Lucifer yellow (1%; Sigma, St. Louis, MA, USA) and the high molecular weight marker dye conjugate Rhodamine B dextran (1%; Sigma, St. Louis, MA, USA) was added to the center region of the coverslip. A 27-gauge needle was used to create longitudinal scratch through the cell monolayer. The cultures were rinsed with PBS 1 min following dye loading. The diffusion of the fluorescent dye was monitored under a laser confocal microscope and the distance of dye diffusion from the scratch margin was measured.

For each imaging time point, Cx3cr1-EGFP or hGFAP-ECFP transgenic mice were anesthetized as described in the previous paragraph and the vascular marker Rhodamine B dextran (M.W. 70 KDa, Sigma-Aldrich, St. Louis, MO, USA) was intravenously injected (3%, 50 μL). Their temperature was monitored and kept at 37 °C using a heating pad during the entire imaging sessions and until their recovery from anesthesia. Animals were head restrained at the stage of a Leica TCS-SP5 2-photon microscope (Leica Microsystems GmbH, Mannheim, Germany) equipped with a 25× water-injection objective (NA 0.9, Leica Microsystems GmbH, Mannheim, Germany) and a Ti-sapphire laser Mai Tai DeepSee (Spectra Physics, Milpitas, CA, USA) tuned to an excitation wavelength of 900 nm. Imaging was performed 100 μm below the dura in the somatosensory cortex. Image z-stacks were typically 1024 × 1024 pixels with a voxel size of 0.144 (x) × 0.144 (y) × 0.988 (z) μm acquired for all samples. A 560 nm dichroic mirror was used to separate 525/50 nm (green channel for EGFP fluorescence detection) or 483/32 nm (cyan channel for ECFP fluorescence detection) and 585/40 nm (red channel for Rhodamine B dextran fluorescence detection) emission filters and fluorescence was collected using NDD detectors.

To determine the lysosomal pH of different cell lines, an in situ pH calibration curve was established following the reported procedure^{19 (link)}. Firstly, Oregon Green-dextran (Invitrogen) and Rhodamine B-dextran (Sigma) were dissolved in DMEM medium at 5 mg mL⁻¹ at a molar ratio of 1: 1. Cell lines were pulsed with mixed fluorescent dextran for 6 h, chased for 12 h in fresh medium, and washed with PBS to calculate the fluorescence intensity of lysosomes by a confocal microscope (A1R-Storm, Nikon) under 60× oil objective lens. Subsequently, a corresponding calibration was performed for each lysosome. The cells were incubated with nigericin (10 μM) in high-K⁺ buffers from pH 4.0 to 6.0 for 5 min equilibrium on the ice. The fluorescence intensity was measured by the confocal microscope. The Hoechst 33342 (Invitrogen), Oregon Green, and Rhodamine B were excited at 405, 488 and 561 nm, respectively. The ratiometric images were processed by NIS-Elements viewer software, and the resulting quantification was calculated by ImageJ software (NIH), which was plotted as a function of pH value and fitted to a Boltzmann sigmoid to measure the lysosomal pH in various cell lines.

Knockdown of ca6 was carried out using two different antisense morpholino oligonucleotides (MOs) (GeneTools LLC, Philomath, OR, USA): one translation-blocking (MO1 5′-CTGCCTGTGCTCTGAACTGTTTCTC-3′) and the other splicing-blocking, to target intron–exon boundary before exon 9 (MO2 5′-GCTTGCCTTGAGAAGGAAAGATCAT). The random control (RC) MOs (5′-CCTCTTACCTCAGTTACAATTTATA-3′) were used as control MOs. The supplied MOs were re-suspended in sterile water at 1 mM stock concentration. Immediately prior to injection, ca6-MOs were diluted to the intended concentration of 125 μM. In order to monitor injection efficiency, 0.2% dextran rhodamine B and 0.1% phenol red (final concentrations; Sigma, Poole, UK) were included in the solution, and the final KCl concentration was adjusted to 1 M. About 1 nl of antisense MO solution was injected into the yolk of approximately 500 one- to two-cell stage embryos, without randomization. The MO-injected embryos were screened for the presence of fluorescence after 24 h to select the true ca6 morphants using Lumar V1.1 fluorescence stereomicroscope (Carl Zeiss MicroImaging GmbH, Göttingen, Germany) and AxioVision software version 4.9. The non-fluorescent embryos were eliminated.