Automatic gamma counter

These parameters were measured as described by Lingelem et al. [28 (link)], with incubation times as specified in the figure legends. For these experiments, ricin was ¹²⁵I-labeled by using Pierce Iodination Tubes (cat. No 28601, Thermo Fisher Scientific, Rockford, IL, USA) according to the protocol from the manufacturer. Ricin endocytosis was measured as the amount of lactose-resistant ¹²⁵I-ricin in percent of total cell-associated ricin as previously described [28 (link)]. The radioactivity was measured using a Hidex Automatic Gamma Counter (Hidex). In principle, degradation of ricin was measured by first incubating with ¹²⁵I-ricin for 20 min, removing the surface-bound toxin with 0.1 M lactose in HEPES-buffered medium, and then chasing the toxin for 2 h in the presence of 1 mM lactose to inhibit rebinding and further uptake of the toxin. At the end of this incubation, the total and the TCA-precipitable radioactivity was measured in the medium, the cells were dissolved in 0.1 M KOH, and the radioactivity in the cells was measured. The pellet from the medium contained the recycled ricin, whereas the non-precipitable radioactivity represents ricin that was degraded during this time-period of incubation. For further details, see [28 (link)].

To evaluate the in vivo biodistribution of Nanosota-1C-Fc and Nanosota-1C, the nanobodies were labeled with Zirconium-89 [⁸⁹Zr] and injected into male C57BL/6 mice (5 to 6 weeks old) (Envigo). Briefly, the nanobodies were first conjugated to the bifunctional chelator p-SCN-Bn-Deferoxamine (DFO, Macrocyclic) as previously described (35 ), and [⁸⁹Zr] (University of Wisconsin Medical Physics Department) was then conjugated as previously described (36 ). [⁸⁹Zr]-labeled nanobodies (1.05 MBq, 1–2 μg nanobody, 100 μl PBS) were intravenously injected (tail-vein). Mice were euthanized at different time points. Organs were collected and counted on an automatic gamma-counter (Hidex). The total number of counts per minute (cpm) for each organ or tissue was compared with a standard sample of known activity and mass. Count data were corrected to both background and decay. The percent injected dose per gram (%ID/g) was calculated by normalization to the total amount of activity injected into each mouse.

¹⁸F-FDG labeling provides a clinically translational approach for PET/MRI tracking of ADSCs. The efficiency of the labeling procedure was investigated for two approaches, microfluidics-based mechanoporation and conventional passive co-incubation. A single-cell suspension of ADSCs (5×10⁶) was mixed with medical-grade ¹⁸F-FDG (57 MBq/ml; 1 ml ¹⁸F-FDG [344 MBq] mixed with 2 ml FACS buffer [3x] and 3 ml PBS) and passed once through the microfluidic device (5 channels, flow rate 0.5 ml/min). We measured the cell processing time through the device for every experiment. Another batch of ADSCs (1×10⁶) was incubated with ¹⁸F-FDG (30 MBq/ml; 0.1 ml ¹⁸F-FDG [33 MBq] mixed with 1 ml glucose-free DMEM) for 12 min at 37°C. The labeled cells were washed twice with PBS to remove residual radioactivity and 5×10⁴ cells were transferred to a 24-well plate for PET imaging (Inveon D-PET, Siemens Preclinical Solutions, Knoxville, TN) using an acquisition time of 10 min and an energy window of 425-650 keV. Unlabeled ADSCs (5×10⁴) were used as control. After PET acquisition, the radioactivity per well was quantified by region of interest (ROI) analysis using the Inveon Research Workplace (IRW) software. Another set of 1×10⁴ labeled cells was measured using an automatic gamma counter (Hidex, Turku, Finland).

Tissues were collected for gamma counting and autoradiography 24 h after injection of [⁶⁴Cu]CD19-mAb as previously described [8 ]. Tissues were collected for both in vitro. Briefly, 200–300 μL of blood was collected from the left cardiac ventricle of anesthetized mice prior to perfusion with 30–40 mL of PBS. Left brain hemisphere, heart, liver, spleen, cervical/thoracic (C/TSC), and lumbar (LSC) spinal cord were harvested, weighed, and counted using an automatic gamma counter (Hidex Oy, Helsinki, Finland), allowing %ID/g to be calculated for each organ of interest. Autoradiography was performed using 40 μm-thick left-brain hemisphere sections and whole C/TSC and LSC to obtain high-resolution images of tracer distribution in CNS tissues, expressed as mean pixel intensity/decay-corrected dose (MPI/dose). Brain section anatomy was confirmed by Nissl staining of the same sections as previously described [15 (link)]. Autoradiography ROIs (n = 4 per group) were drawn on the basis of immunohistochemical findings and were defined as hindbrain (brainstem and cerebellum) and cerebellar white matter (Suppl. Fig. 3B).