Ix81 microscope

Cell proliferation was measured over a period of 4 days after seeding cells at an initial density of 2600 cells/cm². MCF10A NLS copGFP proliferation was continuously monitored by a custom epifluorescence microscope housed in a standard tissue culture incubator (37°C, 90% humidity, 5% CO₂; 10× objective; 1-hour acquisition intervals). Cell numbers were quantified manually every 24 hours using ImageJ. To measure cell viability, cells were seeded at a density of 1300 cells/cm² on fibronectin-functionalized PA and silicone gels in a 12-well plate and assayed after 24 hours with a LIVE/DEAD Viability/Cytotoxicity kit (Thermo Fisher Scientific). Fluorescence images were acquired using GFP and TXRED filter cubes on an Olympus IX81 microscope with a 10× objective (0.25 NA; Zyla 4.2 sCMOS camera) and quantified manually in ImageJ.
Cell proliferation analysis by assaying Ki-67 protein was done as follows: MCF10A cells were seeded at an initial density of 2600 cells/cm², cultured for 48 hours after seeding, and then fixed using 4% paraformaldehyde at room temperature for 30 min. Samples were incubated with Ki-67 primary antibody, followed by Alexa Fluor–conjugated secondary antibody. Nuclei were stained with Hoechst. The number of Ki-67–positive cells and total number of cells were counted manually with ImageJ.

PLBs containing biotinylated anti-mouse Ig surrogate antigen were prepared following our published protocols (40 (link)). B cells were first stained with Alexa Fluor 647 AffiniPure Fab fragment goat anti-mouse IgM, μ chain–specific (115-607-020, Jackson ImmunoResearch Laboratory) and then loaded on the chamber to react with surrogate antigens on the PLBs for at least 10 min, followed by 4% paraformaldehyde (PFA) fixation. TIRFM images were captured using an Olympus IX-81 microscope supported by an Andor iXon⁺ DU-897D electron-multiplying charge-coupled device camera, Olympus 100 × 1.49 numerical aperture objective lens, and TIRF port. TIRFM image acquisition was controlled by MetaMorph software (Molecular Devices), and the exposure time for 512 × 512 pixel images was 100 ms. The mFIs of BCR, FcRL1, and intracellular signaling molecules accumulated at the immunological synapse were statistically analyzed according to the intensity and area using ImageJ software [National Institutes of Health (NIH)].

FL83B cells were seeded in coverslips placed in 12 well plates (150k cells/well). Then, 1.5uM of either Chol-NTC-siRNA or Chol-MCT1-siRNA at final concentration was added for 72 hours. To stain mitochondria membranes, cells were incubated with Mitotracker at 37C (Thermofisher, cat M7512) for 45 min in serum-free media. Then, cells were fixed with 4% paraformaldehyde at room temperature for 30 min. Fixed cells were blocked by fresh permeabilization buffer (0.5% Triton, 1% FBS in PBS) at room temperature for 30 min and incubated with 1:100 Anti-MCT1 (Proteintech, cat 20139-1-AP) overnight at 4C. As a secondary antibody, 1:1000 goat-anti-Rabbit-488 was used, while cells were protected from light. Coverslips were mounted on Prolong Gold antifade Mountant with Dapi (Invitrogen, cat P35934). Images were acquired using an Olympus IX81 microscope (Central Valley, PA) with dual Andor Zyla sCMOS 4.2 cameras (Belfast, UK). Images were quantified using ImageJ software.

TIRFM images were acquired by an Olympus IX-81 microscope equipped with a TIRF port, an Andor iXon⁺ DU-897D electron-multiplying charge-coupled device camera, and an Olympus 100× 1.49 NA objective lens. The acquisition was controlled by MetaMorph software (Molecular Devices). For the imaging options, the exposure time was 100 ms for a 512 × 512–pixel image unless specially indicated. Confocal images were acquired by a FLUOVIEW FV1000 confocal laser scanning microscope (Olympus) with a 60× 1.42 NA oil objective lens. All the images were acquired while keeping the cells in PBS unless specially indicated and were confirmed not to overexpose by the software. All the images were analyzed and processed with ImageJ (National Institutes of Health). The MFI and the total fluorescence intensity (total FI) as AU of BCRs, lipid biosensors, and signaling molecules were calculated based on the intensity analysis as described previously (Lakadamyali et al., 2004 (link); Liu et al., 2010b (link),c (link), 2012 (link)).