Utilizing Bead-Supported Lipid Bilayers to Investigate the Synaptic Output from T Cells

Resuspend 500,000 BSLBs per well in 200 microliters of HBS/HSA buffer. Transfer 100 microliters of BSLBs per well to a new U-bottom 96-well plate to make a duplicate, such that the final amount of BSLB per well is 250,000. Spin down the BSLBs at 300 times g for two minutes at room temperature. Discard the supernatant, and then, resuspend the BSLBs using 100 microliters of T cell suspension. Mix gently to prevent the formation of bubbles. Incubate the co-cultures for 90 minutes at 37 degrees Celsius.

Protect the cells from light, and cool down the co-cultures by first incubating them at room temperature for a minimum of 15 minutes. Centrifuge the co-cultures at room temperature for 5 minutes at 500 times g. Discard the supernatant and resuspend the co-cultures in calcium and magnesium ion-free 2% BSA-PBS at room temperature for blocking. Place the cells on ice for 45 minutes, and protect them from light.

Prepare the antibody master mix using ice-cold 0.22-micrometer-filtered 2% BSA and PBS as a staining buffer. This master mix will provide extra blocking. Spin down the co-cultures at 500 times g for 5 minutes and 4 degrees Celsius. Discard the supernatants, and then, use a multichannel pipette to resuspend the cells in the standing master mix containing optimized antibody concentrations.

Include isotype-labeled cells and BSLBs, fluorescent and non-fluorescent BSLBs, and cells and BSLBs stained alone. Mix gently by pipetting up and down half of the volume. Incubate for 30 minutes on ice and protect them from light. Wash the cells and BSLBs twice using ice-cold 2% BSA-PBS Spin them down at 500 times g for 5 minutes at 4 degrees Celsius. After centrifugation, check the co-cultures sedimented on the bottom of the wells. Then, resuspend the washed co-cultures in 100 microliters of PBS and acquire immediately.