Tmr dextran

For
dextran uptake assay, TZM-bl cells were preincubated with DMSO or
EIPA (50 μM) for 30 min. We added 150 μg/mL tetramethylrhodamine
dextran (TMR-dextran, ThermoFisher Scientific, D1819, 70,000 MW) to
cells and incubated at 37 °C for 30 min.
For transferrin
uptake measurements, TZM-bl cells were pretreated with Dynasore (120
μM), Pitstop2 (20 μM) or DMSO (control) in serum-free
medium. Cells were kept on ice for 5 min, and Transferrin-fluorescein
(Transferrin from Human Serum, Fluorescein Conjugate, ThermoFisher
Scientific, T2871, 50 μg/mL) was added and incubated on ice
for 15 min. Unbound transferrin was removed by two PBS washes, and
the cells were placed at 37 °C for 10 min. EIPA, Pitstop2 or
Dynasore were maintained in medium throughout the experiment (during
preincubation, washing, and postincubation). Cells were washed with
PBS and fixed with 4% paraformaldehyde at 37 °C. Wheat Germ Agglutinin
(WGA) Alexa Fluor 633 Conjugate (ThermoFisher Scientific, W21404)
was used to label the cell membrane. Fluorescence intensity was measured
using a 488 nm laser line for transferrin-fluorescein, a 561 nm laser
line for Dextran-TMR, and a 633 nm laser line for WGA imaging.

[2,3-³H]-l-Serine (specific radioactivity, >5 Ci/mmol) was purchased from Moravek, Inc. (Brea, CA, USA). [2-³H]-Glycine (specific radioactivity, 42.4 Ci/mmol), [3,4-³H]-glutamine (specific radioactivity, 50.5 Ci/mmol) and [³⁵S]-methionine (Specific radioactivity 10.25 mCi/ml) were purchased from PerkinElmer Corp (Waltham, MA, USA). [³H]-glycyl-sarcosine (1.2 Ci/mmol) was purchased from Moravek. SNARF-1-AM and TMR-dextran were purchased from ThermoFisher (Waltham, MA, USA). Niclosamide and pyrvinium chloride were from Sigma-Aldrich (St. Louis, MO, USA).

Glass capillary needles were manufactured using glass capillaries (GC-100TF, Company) and vertical needle puller (model PB-7, Narishige). Solutions were injected into 4hpf zebrafish embryos using Femtojet microinjector (Eppendorf) and Transferman (Eppendorf) micromanipulator mounted to SteroLumar V12 stereomicroscope (Zeiss). The equipment was calibrated using injections of TMR_dextran (0.5 mg/ml in 90% DMSO, 2000 kDa TMR-dextran, Thermo Fischer) into halocarbon oil (Halocarbon oil 27, H8773, Sigma-Aldrich) and in into embryos followed by measurement of fluorescence intensities. Calibration by injections into halocarbon oil indicated delivery of 1.0 +/− 0.06 nl (n = 10), but fluorescence measurement from the embryos indicated that actual amount delivered in vivo was 2.0 +/− 1.2 nl (n = 10). In the toxicity experiment, either DMSO alone or MSNs dissolved in DMSO (10 mg/ml) were injected. After injections, damaged embryos were removed and healthy embryos were cultured in E3 at 28.5C.

Vero cells that were pretreated with the indicated inhibitors or transiently expressed dominant-negative RhoA, Cdc42, Rac1, or Pak1 were pulsed for 20 min with the fluid-phase marker 70 kDa TMR–dextran (1 mg/ml; Thermo Fisher Scientific). Surface-bound dextran was removed with a low pH wash (0.1 M sodium acetate, 0.05 M NaCl, pH 5.5) prior to formaldehyde fixation. Internalized TMR–dextran was analyzed by confocal laser scanning microscopy.

Fast Blue (Polysciences, 17740-1), CM-DiI (Thermo Fisher, C7000), Fast DiI (Thermo Fisher, D3899), TMR-Dextran (Thermo Fisher, D3308), FluoroGold (Fluorochrome) as candidate fluorescent tracers were scanned for their compatibility with the dehydration protocol of laser capture microdissection before the tracer was adapted in the minipig. In each animal, one of the five tracers was injected into Sprague Dawley rats (3-4 months old, purchased from Envigo) sino-atrial node at the same volume of 10 μL and the heart tissues were harvested in the time window 10 am-12 pm 14 days after injection and stored in OCT immediately. Cryosections were visualized under a fluorescence microscope before and after the dehydration steps necessary for laser capture microdissection and acquisition of single neuron samples (as detailed below). All five tracers labeled intrinsic cardiac nervous system neurons successfully but only CM-DiI and Fast Blue-labeled neurons remained intact on the heart sections without fixation, whereas TMR-Dextran and FluoroGold-stained neurons were not visible under the microscope under the conditions suitable for laser capture microdissection. Only Fast Blue provided reliable and consistent labeling visible under laser capture microdissection microscope after the necessary dehydration procedure and was used in the subsequent tracing experiments in the study.