Imagestream x mark 2 flow cytometer

The leukemia cell line, Jurkat (ATCC TIB-152), and prostate cancer cell line, LnCap (ATCC CRL-1740), were obtained from the ATCC (Rockville, MD, USA). Both cell lines were grown under standard cultivation conditions, 37°C, 5% CO₂ in RPMI1640 medium supplemented with 10% fetal bovine serum. The cells were incubated with scFv clone (H5 or G2-H7), anti-Ku80 mouse antibody 5C5 (Abcam, Cat. no ab119935), anti-Tn antigen antibody GOD3-2C4 [21 (link)], non-binding scFv clone or mouse IgG1 as negative controls. The scFv clones were detected with a mouse anti-His antibody, and polyclonal allophycocyanin-conjugated anti-mouse IgG antibody (Jackson ImmunoResearch Labs, Cat. no 115-136-146). The scFv clone H5 targeting peptide 6 was analysed with flow cytometry using a BD FACSAria II, cell sorter (BD Biosciences), using Sytox® Green nucleic acid stain (Invitrogen) as viability stain. The scFv clone G2-H7 was analysed with an ImageStream®x Mark II flow cytometer (Merck).

Cells were fixed in 4% paraformaldehyde (10 min), permeated with 0.2% Triton X-100 (15 min), and blocked with 1% bovine serum albumin (30 min). The anti-FGFR1 primary antibody (ab824, Abcam) was incubated for 90 min, and the AlexaFluor 647 goat anti-rabbit (A27040, Invitrogen) secondary antibody for 45 min. Then, cells were incubated with DAPI (Sigma) for 10 min to stain nuclei. A filter of 30-µm was used to remove cell aggregates. ImageStream^X Mark II Flow Cytometer (EMD Millipore) was used to acquire single cells images at 60× magnification. The acquired raw image file (.rif) contained among 500 and 2000 events (10–30 events per second). The analysis of single cells fluorescence intensity and nucleus diameter was performed by using IDEAS software (version 6.2.64.0). To consider only single cells, a dot plot showing area versus aspect ratio (AR) was created. To estimate FGFR1 intensity in nuclear region, we generated a morphology mask that defined nucleus stained by DAPI and to measure the fluorescent signal in nuclear area.