Goat anti rat igg alexa647

Flow cytometry was carried out to examine the cellular affinity of antibodies and anti‐TF1859‐NC‐6300 for BxPC3 and SUIT2. BxPC3 and SUIT2 were washed with PBS and detached by incubation with a non‐enzymatic cell dissociation solution (×1) for 15 min at 37°C. Suspensions of cells with B.E. PBS were put into flow cytometry‐compatible tubes (BD Biosciences, Franklin Lakes, NJ, USA) at 2 × 10⁵ cells/tube. Suspensions were then centrifuged at 500 g for 5 min, and the supernatant was removed. The cells were incubated for 30 min on ice with primary antibodies and anti‐TF1859‐NC‐6300 (equivalent to 0.5 μg mAb for 1 × 10⁶ cells) diluted in B.E. PBS. Then, the cells were washed with B.E. PBS and incubated for 30 min on ice with goat anti‐rat IgG‐Alexa647 (Invitrogen, Grand Island, NY, USA) as a secondary antibody diluted in B.E. PBS. Controls contained only secondary antibody without primary antibody. After washing and staining with propidium iodide, the cells were applied to a flow cytometer (Guava EasyCyte; GE Healthcare). Multi‐parametric analyses were carried out using FlowJo 7.5.5 software (Ashland, OR, USA).