Millicell ez slide eight well glass

Cell migration chambers (Millicell EZ slide eight-well glass, Millipore) were prepared by coating with Protein A (Invitrogen, 20 μg/mL), ICAM-1 (2.75 μg/mL), CXCL10 and CXCL12 (400 ng/mL) in PBS. 5x10⁴ PD1-sorted CD8⁺ T cells were plated in Leibovitz’s medium supplemented with 2 mg/mL D-glucose in a 37°C chamber. Video microscopy was conducted using a TE2000-U microscope (Nikon) coupled to a CoolSNAP HQ CCD camera with a 10x objective and 0.45 numerical aperture. T Cells and cancer cells were stained with CellTrace CFSE, CellTrace Violet, or CellTrace FarRed per manufacturers protocol (ThermoFisher). T cells were plated in L-15 medium at least 20 min at 37°C before imaging. For T cells alone, bright field or DIC images were acquired every 15 sec for 20 min. For T cell and cancer cell coculture movies, the stained cancer cells were plated 24 hours before the start of imaging. Bright field and fluorescent images were captured every 30 seconds for at least 2 hours.

Cell migration chambers (Millicell EZ slide eight-well glass, Millipore) were prepared by coating with Protein A (20 μg/mL), ICAM-1 (2.75 μg/mL), and CXCL12 (400 ng/mL) in PBS. Day 4 activated CD8⁺ T cells were plated in L-15 medium (Invitrogen, Leibovitz’s medium + 2 mg/mL D-glucose) in a 37°C chamber. Video microscopy was conducted using a TE2000-U microscope (Nikon) coupled to a CoolSNAP HQ CCD camera with a 10x objective and 0.45 numerical aperture. T cells were plated in L-15 medium at least 20 minutes at 37°C before imaging. Treatment of cells with inhibitors before imaging: CoCl₂ treatment was overnight (~16 hours) and maintained in L-15 media; FCCP and oligomycin were added to the cells 20 minutes before the movie started when cells are plated. OptoMito-On and Mock or GFP T cell migration movies all received 500 nm illumination with a Texas Red filter. Bright field or DIC images were acquired every 15 seconds for 20 minutes.

Immunofluorescence staining was performed as previously done (17 (link)). Briefly, 5 * 10⁴ cells were seeded in each well of Millicell EZ SLIDE eight-well glass (Millipore), fixed with 4% PFA, and permeabilized with 0.1% Triton X-100. After blocking with 2.5% goat serum, the cells were incubated with rabbit anti-human estrogen receptor α antibody (D8H8, 1:1,600, CST), rabbit anti-human progesterone receptor A/B (D8Q2J, 1:800, CST), or rabbit anti-human HER2/ErbB2 (D8Q2J, 1:400, CST) overnight at 4°C. After rinsing with phosphate-buffered saline (PBS), anti-rabbit IgG (Alexa Fluor 555 conjugate, CST) was used to incubate the cells for 30 min. Alexa Fluor 555-conjugated rabbit IgG (Alexa Fluor 555 conjugate, CST) was used as the negative control. The cells were then rinsed with PBS three times and stained with DAPI (CST) for 1 min. A Zeiss Axiocam fluorescence microscope was used for image acquisition and analysis.