Transduction was performed as previously described.20 (link) Briefly, non-tissue culture-treated plates were coated with RetroNectin (5 µg/mL; Takara). On the following day, the plates were blocked with 2% bovine serum albumin-phosphate-buffered saline (PBS), loaded with CAR4 or EV retroviral particles, and centrifuged at 3300 rpm for 2.5–3 hours at 30°C, and the supernatants were discarded. DNTs expanded for 3–4 days were resuspended at a concentration of 0.25×106 to 0.4×106 cells/mL in RPMI 1640 medium supplemented with 10% FBS, IL-2 (250 IU/mL), and anti-CD3 antibody (0.05 µg/mL; OKT3) and added to the viral loaded plates. DNTs were split on day 3 after transduction and every 3–4 days thereafter, where fresh RPMI 1640 medium supplemented with 10% FBS, IL-2 (250 IU/mL), and soluble anti-CD3 antibody (0.1 ug/mL; OKT3) were added. DNTs were harvested 10–20 days post expansion for subsequent experiments. To evaluate the level of CAR4 expression, cells were stained with biotinylated protein L antibody (3 µg/100 µl; Thermo Fisher Scientific), followed by phycoerythrin-conjugated streptavidin (2 µg/100 µl; BioLegend), and analyzed by flow cytometry.
Phycoerythrin conjugated streptavidin
Manufactured by BioLegend
Sourced in United States
Phycoerythrin-conjugated streptavidin is a fluorescent protein-based reagent. It consists of the protein streptavidin, which has a high affinity for biotin, conjugated to the fluorescent dye phycoerythrin. This product can be used to detect and visualize biotinylated molecules in various applications, such as flow cytometry and fluorescence microscopy.
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2 protocols using phycoerythrin conjugated streptavidin
1
Transduction and Expansion of CAR-T Cells
2
Rat Alveolar Macrophage Isolation and Characterization
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Four naïve rats were anesthetized under isoflurane anesthesia and bronchoalveolar lavage (BAL) was performed with 2 mL of PBS. The BAL fluid was centrifuged at 2,000 ×g, the resulting cell pellet was resuspended in FACS staining buffer, and Fc receptors were blocked for 30 min at 4°C. Cells were stained for 30 min with an Alexa Fluor 700 anti-rat CD45 antibody (BioLegend, San Diego, CA, United States), an Alexa Fluor 488 mouse anti-rat CD11b antibody (Bio-Rad, Hercules, CA, United States), a mouse anti-rat CD11c antibody (Bio-Rad) pre-conjugated with Cy5, and a biotin-conjugated mouse anti-rat CD68 antibody (Bio-Rad), followed by a phycoerythrin-conjugated streptavidin (BioLegend). Finally, all cell samples were stained with DAPI. Cell populations were analyzed using a BD Influx Cell Sorter (BD Biosciences, San Jose, CA, United States), and flow cytometry compensation setting was performed with UltraComp eBeadsTM (Thermo Fisher Scientific, Waltham, MA, United States) and with cell samples. The NR8383 rat alveolar macrophage cell line was used as a positive control for staining and flow cytometry.
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