Scintiverse

Neurotransmitter uptake
assays were performed as previously reported.^{59 (link),94 (link)} Briefly, cells were washed with room temperature phosphate buffer
PBS-CM (2.7 mM KCl; 1.2 mM KH₂PO₄, 138 mM NaCl;
8.1 mM Na₂HPO₄, added 0.1 mM CaCl₂ and 1 mM MgCl₂, pH 7.4) and incubated for 10 min at 37
°C with compounds (R)-7 [(R)-AS-1] and (R)-8 [(R)-AS-7] at 0.01–100 μM final concentration. Uptake reactions
were initiated by the addition of an appropriate radiolabeled substrate
([³H]-l-glutamate or [³H]-GABA) at
a final concentration of 50 nM. After 10 min, reactions were terminated
in two washes with buffer and lysis with 1% sodium dodecyl sulfate
(SDS)/0.1 M NaOH. The lysate was transferred to scintillation vials
containing 3 mL of scintillation fluid (ScintiVerse, Fisher Scientific,
Pittsburgh, PA) and radioactivity was quantified in a scintillation
counter LS 6500 (Beckman Coulter, Brea, CA).

Caco-2 cells at 10 days postseeding were incubated with 10 µmol/L Gly-Sar (9.98 µmol/L Gly-Sar and 0.02 µmol/L [³H]Gly-Sar (Moravek Biochemicals, Brea, CA, USA)), along with various concentrations (5–0.05 mmol/L) of Gem and Gem prodrugs for 30 min. After the incubation, the drug solution was removed. The cells were gently washed three times with ice-cold PBS and solubilized with 2 mL of scintillation cocktail (ScintiVerse, Fisher Scientific, St. Louis, MO, USA), and the radioactivity was determined by scintillation counting (Beckman LS-9000, Beckman Instruments, Fullerton, CA, USA). Inhibitory concentration 50 (IC₅₀) values were determined using nonlinear data fitting (GraphPad Prism version 6, GraphPad Software, Inc., La Jolla, CA, USA).

A 0.5 mL aliquot of the above-described hexane extract was evaporated to dryness in a water bath under a gentle flow of nitrogen, then used for determination of ³H tracer by liquid scintillation spectrometry (Beckman Coulter, Brea, CA, USA) using Scintiverse (Fisher Chemical, Waltham, MA, USA) as scintillation fluid, as described earlier [35 (link)]. Samples were counted to a 2-sigma error of 1% or for a maximum of 60 min.
To further identify the form of VA in intestine, we separated retinyl esters (RE) and retinol from the hexane extract using alumina column chromatography [36 (link)]. Briefly, the hexane extract was loaded onto the column, and then the column was sequentially eluted with 3% diethyl ether and 50% diethyl ether to obtain the retinyl ester- and retinol fractions, respectively. Then, ³H content in these fractions was determined as mentioned above.

HSV-1- or EIV-specific responder cells were quantified by a modified limiting dilution lymphoproliferation assay (55 (link)), using either cell-free HSV-1 or EIV Prague/56 HA protein lysate and control antigen lysate. Briefly, wells of 96-well plates were coated with 20 μl/well of antigen extract (HSV-1 or RS cell control lysate, or EIV Prague/56 HA or Vero cell control lysate) and were allowed to air dry in a laminar flow hood. Dilutions of mouse PBMCs in DMEM were added to each well so that each well contained a minimum of 1 and a maximum of 10 lymphocytes per well in a volume of 100 μl complete medium (with serum). The plates were then incubated at 37°C. After 24 h, medium containing 10 μCi of [³H]thymidine was added to each plate for 12 h, the medium was replaced, and the plates were incubated for an additional 72 h. The wells were harvested, and the DNA was precipitated in 20 volumes of cold 10% trichloroacetic acid, transferred onto glass fiber discs (Whatman; GF/C) by filtration, rinsed with 95% ethanol, and dried using a heat lamp. The filters were then transferred to scintillation vials with Scintiverse (Fisher Scientific) and counted. The counts per minute (cpm) of [³H]thymidine were converted to RCF using the maximum-likelihood estimate method of Levin et al. (56 (link)).