Mcl 400

After 6 weeks of incubation post FACS-sorting, six clones from the 96-well plate exhibited cell growth. These clones were trypsinized and expanded into a T25 flask (Corning; 430639) and incubated at 37 °C with 5% CO₂ humidity for an additional 2 weeks of growth. To validate the integration of the biotin tag into PSMD2, live cells expressing mScarlet were visualized by confocal microscopy. Imaging was performed by an Eclipse Ti inverted microscope (Nikon Inc) equipped with a Yokogawa spinning disc (Yokogawa Electric Corporation), back-illuminated EMCCD camera (Andor, DU888), and a 2x relay lens placed before the spinning disc. A 60x, NA 1.45, Plan Apo objective and a 561 nm laser (Agilent Technology MCL-400) was used to acquire fluorescence (mScarlet) images in parallel with differential interference contrast images for reference.

All live imaging were interchangeably carried out, based on requirement, in one of the following setups: 1) confocal spinning disk microscope (for imaging blebs in 3D) equipped with a Yokogawa CSU-22 unit and 100×, 1.4NA Nikon oil objective; Andor technologies laser combiner emitting 488 and 561 nm wavelengths, amongst others; and Andor ixon+897 EMCCD cameras. Images were acquired using Andor iQ2 software. 2) Total internal reflection fluorescence (TIRF) microscope setup was equipped with Nikon Eclipse Ti body; a 100×, 1.45NA Nikon oil objective; photometrics Evolve EMCCD cameras; an Agilent laser combiner MCL400 (Agilent Technologies) whose 488, 561, and 640 nm excitation wavelengths were used as necessary; and µManager for image acquisition. 3) TIRF microscope setup was equipped with Nikon TE2000 body; a 100×, 1.49NA Nikon oil objective; EMCCD Cascade 512 cameras (Photometrics, Tuscon, AZ); a home-built laser combiner equipped with 488 and 561 nm lasers; and Metamorph/µManager for image acquisition. Wherever necessary, live imaging was performed in a temperature-controlled stage-top incubator chamber with immersion thermostat, ECO Silver, from Lauda Brinkmann.

Cells were plated on coverslips and transfected with a plasmid encoding mCherry-EB3 (a kind gift from Dr. Irina Kaverina, Vanderbilt University, Nashville, TN) using GenJet (SignaGen Laboratories) following the manufacturer’s instructions. ∼24 h after transfection, cells were mounted in Attofluor Cell chambers (A7816; Thermo Fisher Scientific) and imaged using an Eclipse Ti inverted microscope (Nikon) equipped with a Yokogawa spinning disc (Yokogawa Electric Corp.), 405-, 488-, 561-, and 640-nm laser launch (MCL-400; Agilent Technology), and a back-illuminated EMCCD camera (DU888; Andor), 100×, NA 1.42, Plan Apo objective, with a 2× relay lens placed before the spinning disc. A time-lapse over 1 min was recorded imaging one z plane each 1 s, using 200-ms exposure time. 4-s time projections were generated in Fiji (National Institutes of Health). The length of the comet over a 4-s period was determined if the same comet appeared over more than one 4-s timeframes. The displacement of the EB3 comets in 1 s was calculated and plotted.