Wallac wizard 1470 020 gamma counter

The cell-based ¹²⁵I-GDNF-displacement assay
was carried
in HEK293 cells. The cells were cultured in DMEM, 10% FBS, and 100
μg/mL of normocin overnight and then transfected with GFRα1
or GFRα1 and RET using Lipofectamine 2000 (Invitrogen), as described
by the manufacturer. The next day, the cells were incubated for 1
h on ice in the presence of the BT44 formulation in concentrations
ranging from 0 to 50 pM. Afterward, 50 pM of iodinated GDNF was added,
and the cells were incubated for 1 h on ice with ¹²⁵I-GDNF
and nanoformulated BT44. Subsequently, cells were washed with PBS
four times and lysed with 1 M NaOH, and lysates were collected into
the scintillation vials and counted using a PerkinElmer Wallac Wizard
1470-020 Gamma Counter. The data for GFRα1-transfected cells
was collected in two independent experiments and for GFRα1/RET-transfected
cells in four independent experiments. The binding data were analyzed
by nonlinear regression analysis using GraphPad Prism 8.4.2 software
to determine the IC₅₀ values. Experiments with nonformulated
BT44 were not conducted because of insufficient solubility of the
compound in assay media.

The intestinal permeability (IP) can assess gut mucosa integrity. A 0.1 mL (18.5 MBq) sample of diethylenetriamine penta-acetic acid (DTPA) solution labeled with technetium-99m (99mTc-DTPA) was administrated by gavage to the mice on the 13th day of treatment, as previously reported [53 (link)]. Four hours later, the mice were anesthetized as previously described. Approximately 200 µL blood was collected, weighed, and the radioactivity level in the blood was determined in an automated gamma counter (PerkinElmer Wallac Wizard 1470-020 Gamma Counter, Waltham, MA, USA). A standard dosage containing the same injected amount was counted simultaneously in a separate tube, which was defined as 100% radioactivity. The results were expressed as the percentage of injected dose per gram (%ID/g) of blood: counts per min = (cpm of blood/cpm of the administered dose) × 100. where cpm represents the count of radioactivity per minute.

Intestinal permeability was determined by measuring radioactivity diffusion in the blood after oral administration of diethylenetriamine penta-acetic acid (DTPA) labeled with 99 m-technetium (99mTc) as previously described (23 (link)). After 7 days of diet consumption, animals received 0.1 mL of a DTPA solution labeled with 18.5 mebequerel (MBq) of 99mTc by gavage. Four hours later, mice were anesthetized, their blood was collected, weighed, and placed in appropriate tubes for radioactivity determination. Blood radioactivity levels were determined using an automated gamma counter (Perkin Elmer Wallac Wizard 1470-020 Gamma Counter; PerkinElmer Inc., Waltham, MA, USA). Obtained results were compared with the standard dose and calculated as a percentage of the dose per gram of blood using the following equation:
$\text{\%}\text{dose}/\text{g blood}=\left(\begin{array}{l}\text{cpm in g of blood}/\\ \text{cpm dose of standard}\end{array}\right)\times 100,$
where cpm represents the counts of radioactivity per minute.

Mice received 0.1 mL of DTPA (diethylenetriaminepentaacetic acid) via gavage, labeled with 18.5 MBq 99mtechnetium on the last experimental day (de Barros et al., 2018 (link)). After four hours, total blood was collected without any stabilizer and measured the radioactivity level in an automated gamma radiation counter (PerkinElmer Wallac Wizard 1470-020 Gamma Counter, Waltham, MA, United States). Permeability was calculated by the percentage of DTPA administered dose per gram of blood (% ID/g), as follows: counts per min = (blood cpm/cpm of the administered dose) × 100.

Intestinal permeability was determined by measuring radioactivity diffusion in the blood after oral administration of diethylenetriamine pentaacetic acid (DTPA) labeled with ^99m-technetium (^99mTc). After 21 days of diet consumption, animals received 0.1 mL of a DTPA solution labeled with 18.5 mebequerel of ^99mTc by gavage. Four hours later, mice were anesthetized; their blood was collected, weighed, and placed in appropriate tubes for radioactivity determination. Blood radioactivity levels were determined using an automated gamma counter (Perkin Elmer Wallac Wizard 1470–020 Gamma Counter; PerkinElmer Inc., Waltham, MA, USA). Results obtained were compared with the standard dose and calculated as a percentage of the dose per gram of blood using the following equation:
$\%\text{ dose/g blood}=\left(\text{cpm in\hspace{0.17em}g\hspace{0.17em}of blood/cpm dose of standard}\right)\times 100,$
where cpm represents the counts of radioactivity per minute.