Lsm 510 meta confocal module

For live-cell imaging, HL-60 neutrophils were collected, washed and resuspended with mHBSS containing 0.2% BSA, and then seeded on Lab-Tek chambered cover glasses (NUNC) coated with 50 μg/ml human fibronectin (Sigma-aldrich). Time-lapse microscopy was performed on a Leica Spinning Disk Confocal microscope. Chemoattractant fMLP (Sigma) was given to cells either globally or delivered by a Femtotips micropipette (Eppendorf). Total Internal Reflection Fluorescence (TIRF) microscopy was performed using a Nikon Eclipse Ti-E TIRF microscope. Fluorescence recovery after photobleaching (FRAP) was performed on Zeiss Axiovert 200 Laser scanning microscope with LSM510-Meta confocal module. For immuno uorescent staining, cells were xed, and then stained with Anti-PI 3-Kinase, p110γ (clone 17D7.2, Merck Millipore) in PBS containing 1% BSA as indicated in the Extended Experimental Procedures. Images were analyzed with ImageJ (NIH) as described in the Extended Experimental Procedures.

Internalization and localization of 5D3 mAb and its drug conjugates were studied in PSMA(±) cells. PSMA(+) PC3-PIP or PSMA(–) PC3-Flu cells were seeded (4-well chamber slides, 0.2 million cells per well) and grown for 1–2 days to 80–90% confluency. Medium was removed in each chamber, and cells were treated with 150 μL of 20 μg/mL of 5D3-AF-488 or 5D3-DM1-AF-488 and incubated at 37 °C for 30 min, 1, 6, 12, and 24 h under 5% CO₂ in a humidity-controlled incubator. For one chamber treated with 5D3-AF-488, cells were pulsed with 70 kDa rhodamine–dextran (100 μg/mL) along with 5D3-AF-488. Cells were washed with DPBS for 5 min and fixed by 4% PFA on ice for 10 min. Nuclei were counterstained using Hoechst 33342 (10 μg/mL in H₂O at RT for 10 min) and wet-mounted. The images were collected using a Zeiss Axiovert 200 fluorescence microscope system equipped with an LSM 510-Meta confocal module and processed using Zeiss Zen software.