The medium was then removed from the cells and 100 mL of 1X dye binding solution was added and plates were incubated for one hour at 37°C.
Briefly, the cell growth medium was removed and 100 µl of 1x lysis/dye binding solution was added into each well.
After incubation, 400 μl binding solution was added to each tube and fluorescence was detected using flow cytometer (BD Biosciences).
For IgG1 binding assays, anti-HA IgG1 in blocking solution were added to wells and incubated for 1 hour at room temperature, after which bound IgG1 was detected using a peroxidase-conjugated mouse anti-human IgG antibody (Jackson).
Without washing, another 200 microliters of binding buffer and 5 microliters of PI solution were added, and 800,000 cells were acquired by flow cytometry (FACSCalibur, Becton Dickinson) and analyzed by CellQuest software (BD Bioscience).
ARPE-19 cells were resuspended in 200 μl Annexin binding buffer at 200 × 10 cells/ml, V-FITC and PI solution were added.
The solutions i.e. QC samples, CC standards and unknown plasma samples, were freshly prepared as; each sample (600 µl aliquot) alongwith a 50 µl IS (50 ng/ml) was taken in a glass tub, 5% formic acid (200 µl) solution was added (breaking protein binding) and vortexed (300 rpm, 5 min).
After mixing with 240 μl of NaClO4 binding buffer, 60 μl of glass milk solution was added and the suspension was gently shaken at room temperature for 10 min then centrifuged at 7,000 × g for 30 s.
Then Gd3+ (as Gd3+ in solution) was added to preformed lipid nanoparticles (d = 60 70 nm) expressing DTPA-PE (for binding to Gd3+ as Gd-DTPA-PE chelate).
