Eclipse ti inverted microscope

All imaging in this study was performed using a spinning disk confocal microscope with environmental control (Nikon Ti-Eclipse inverted microscope with a Yokogawa spinning disk unit). For bead internalization assays, images were acquired using a 40 × 0.95 N/A air objective and unbiased live image acquisition was performed using the High Content Screening (HCS) Site Generator plugin in µManager³. Other images were acquired using either a 100 × 1.49 N/A oil immersion objective. All images were acquired using an Andor iXon EM-CCD camera. The open source µManager software package was used to control the microscope and acquire all images³.

Images were acquired on a spinning disc confocal microscope (Nikon Ti-Eclipse inverted microscope with a Yokogawa CSU-X spinning disk unit and an Andor iXon EM-CCD camera) equipped with a 40 × 0.95 NA air and a 100 × 1.49 NA oil immersion objective. The microscope was controlled using µManager. For TIRF imaging, images were acquired on the same microscope with a motorized TIRF arm using a Hamamatsu Flash 4.0 camera and the 100 × 1.49 NA oil immersion objective.

Cells were seeded on 24-well plates on coverslips pre-treated with poly-lysine. HEK293T cells expressing a control guide RNA targeting the LacZ gene or a guide RNA targeting ZAP were transfected with 250 ng of pKHNYN-FLAG. 24 hr post-transfection, the cells were fixed with 4% paraformaldehyde for 15 min at room temperature, washed with PBS, and then washed with 10 mM glycine. The cells were then permeabilized for 15 min with 1% BSA and 0.1% Triton-X in PBS. Mouse anti-FLAG (1:500) and rabbit anti-ZAP (1:500) antibodies were diluted in PBS/0.01% Triton-X and the cells were stained for 1 hr at room temperature. The cells were then washed three times in PBS/0.01% Triton-X and incubated with Alexa Fluor 594 anti-mouse and Alexa Fluor 488 anti-rabbit antibodies (Molecular Probes, 1:500 in PBS/0.01% Triton-X) for 45 min in the dark. Finally, the coverslips were washed three times with PBS/0.01% Triton-X and then mounted on slides with Prolong Diamond Antifade Mountant with DAPI (Invitrogen). Imaging was performed on a Nikon Eclipse Ti Inverted Microscope, equipped with a Yokogawa CSU/X1-spinning disk unit, under 60-100x objectives and laser wavelengths of 405 nm, 488 nm and 561 nm. Image processing and co-localization analysis was performed with NIS Elements Viewer and Image J (Fiji) software.