Countess 3

For determining the doubling time of cells expressing RNA exporters, as shown in Figure S4B, HEK293T cells were plated on 96-well plates with 8,000 cells per well. Cells were co-transfected the following day with 100 ng of RNA exporter plasmid (30 ng in the case of EPN24-MCP and EPN11-MCP) using Lipofectamine 3000 (ThermoFisher) according to manufacturer’s instructions. At 24 and 48 hours after transfection, media was removed, cells were lifted from the plate by adding 50 μL of Trypsin-EDTA (ThermoFisher) and incubating at 37 C for 5 minutes, and cells were resuspended by adding 50 μL of media. Microscopy confirmed that no cells remained attached to the plate. Cell counts were determined using the Countess 3 automated cell counter (ThermoFisher). Doubling time T_d was calculated using the equation T_d = Δt / log2(N₂/N₁), where Δt is the time difference between the timepoints (24 hours), N₁ is the cell count at 24 hours, and N₂ is the cell count at 48 hours. Three replicate wells were counted for each sample at each timepoint and their variance was propagated to the estimate of T_d. Cells remained sub-confluent at 48 hours after transfection and exhibited stable expression of transfected plasmids based on fluorescent protein markers at both 24 and 48 hour timepoints.

To determine UV intensity, 2.5 × 10⁵ PF1 cells in 500 µl media were plated in six-well dishes. UVC treatments used the Analytik Jena crosslinker (model CL-1000, 254 nm) at exposures of 5,000–30,000 µJ/cm² (Extended Data Fig. 2a). After treatment, 2 ml fresh media was added and cells cultured for 3 days. Cell numbers were counted in triplicate using the Countess 3 from Thermo Fisher. UV at 5,000 µJ/cm² was used given roughly half of the cells proliferated post treatment.

This study was conducted at the Zhongdi Dairy Research Center in Beijing, China. In the experiment, all cows were housed in the same barn and had access to a total mixed ration (TMR) with a forage-to-concentrate ratio of 60:40 and water ad libitum. The TMR was added three times a day (0,700, 1,430, and 2,200), and its formulation complied with NRC (2001) requirements. The composition and nutritional information of the TMR are shown in Table 1.
The study included a total of 23 Holstein cows, with 10 in the healthy group (H group) and 13 in the clinical mastitis group (M group). Clinical mastitis in cows was diagnosed by an experienced veterinarian. Initially, cows with recent decreases in milk production were selected using dairy management software (Valley Ag software, PA, United States). Subsequently, the veterinarian assessed the udders for clinical symptoms such as swelling, heat, and hardness using palpation in the milking parlor. Finally, milk samples from cows exhibiting clinical symptoms were collected and subjected to SCC testing using an instrument (Countess 3, Thermo Fisher, Waltham, United States), with 500,000 cells/mL set as the threshold for clinical mastitis, thus confirming cases of mastitis in the cows. Healthy cows were identified based on criteria such as normal milk production and udders free from abnormalities.

5 × 10⁵human T cells were infected by the lentivirus mentioned above. To draw cell growth curves, live T cells were counted using trypan blue staining and Automated Cell Counter (Thermo Fisher, Countess™ 3). Expression of CD19-, BCMA-, BC19- or 19BC CAR was evaluated by Biotinylated protein L (Acro Biosystems, Cat: RPL-P814R, Lot: BL11R-76EF1-GY,1:400 dilution) and APC--conjugated Streptavidin (Biolegend, Cat: 405207, Lot: B388667, 1:100 dilution). PE-labeled anti-CD45RA (Biolegend, Clone: HI100, Cat: 304107, Lot: B378521, 1:100 dilution) and PE-Cy7-labeled anti-CD62L (Biolegend, Clone: DREG-56, Cat: 304821, Lot: B373156, 1:100 dilution) were employed to determine phenotypes of infusion products of CAR T, including naïve (CD45RA⁺ CD62L⁺), central memory (CD45RA⁻ CD62L⁺), effector memory (CD45RA⁻ CD62L⁻), and effector (CD45RA⁺ CD62L⁻) T cells.

Cells were seeded in 24-well plates, and 1 h before [⁶⁸Ga]KISS1-54 uptake, the cell culture medium was replaced by PBS/5% BSA. Cells were incubated at 37 °C with [⁶⁸Ga]KISS1-54 for 5–90 min or co-incubated for 60 min with [⁶⁸Ga]KISS1-54 and KISS1-54 (Sigma Aldrich, Deisenhofen, Germany). After incubation, cells were washed three times with ice-cold PBS/5% BSA and harvested. Radioactivity was measured with a gamma-counter (Wizard 2480, Perkin-Elmer, Rodgau, Germany), and cells were subsequently counted with Countess 3 (Thermo Fisher Scientific, Waltham, MA, USA). In the case of treatment with 5-Aza-2′-Deoxycytidine (Decitabine (DC)), cells were preincubated for 72 h in cell culture medium containing 5 µM Decitabine (Merck Millipore, Darmstadt, Germany).
For data analysis, the determined accumulation of [⁶⁸Ga]KISS1-54 in vitro was standardized as “uptake/100,000 cells” for a better comparison. For example, the cell number differed between pretreatment with and without Decitabine, which is due to the treatment itself, making standardization necessary.

Neural progenitor cells (NPCs) were created from the hESCs described above using the STEMdiff SMADi Neural Induction kit from STEMCELL Technologies (Cat No. 08581) according to their guidelines for Matrigel-coated 6-well plates [31 ]. Briefly, colonies were checked for areas of differentiation and removed, if necessary, before washing once with cold DPBS-/-. Colonies were then incubated for 8–10 min at 37 °C in GCDR for dissociation. Cells were collected and triturated to create a single-cell suspension, then spun down at 300 g for 5 min at room temperature. Cell counts were determined using a Countess 3 instrument (Cat No. AMQAX2000, Thermo Fisher), and 2,000,000 cells/well were plated in a 6-well plate in STEMdiff SMADi Neural Induction media. Daily media changes were performed for seven days followed by passaging of progenitor cells using Accutase (Cat No. 07920, STEMCELL Technologies), which was repeated for one additional passage (two passages total). Cells were plated at 1,500,000 cells/well in a 6-well plate for each passage. At passage 3, cells were either frozen down in CryoStor CS10 (Cat No. 07930, STEMCELL Technologies) at 3,000,000 cells/vial or moved onto the next step of the differentiation process.

Cell viability was measured by Trypan Blue staining assay. 0.4% Trypan Blue solution was added to a single-cell suspension at a ratio of 1:9 in volume. Live/dead cells were read using Invitrogen Countess 3 (ThermoFisher) and the percentage of live cells was calculated.