This article discusses the use of bead-supported lipid bilayers (BSLBs) as synthetic antigen-presenting cells to study T cell activation. The methodology involves incubating T cells with BSLBs to analyze the formation of immune synapses and subsequent signaling events.
Take bead-supported lipid bilayers, or BSLBs, silica beads coated with lipid bilayers conjugated with antigenic peptide-MHC complexes, adhesion and co-stimulatory molecules, acting as synthetic antigen-presenting cells.
Incubate with T cells.
TCRs bind to antigenic peptides on BSLBs, which, along with co-stimulatory signals, form an immune synapse, activating T cells.
This binding triggers intracellular signaling cascades, inducing actin rearrangement and forming microclusters at the synapse containing adhesion and signaling molecules.
T cells release trans-synaptic vesicles enriched in TCRs, co-stimulatory molecules, and lytic granule contents into the synapse, binding to BSLBs.
Gradually cool down the co-culture to separate BSLBs from T cells.
Centrifuge and discard the supernatant. Use a protein-containing buffer to block the free binding sites on the surfaces.
Centrifuge and treat BSLBs and T cells with fluorophore-conjugated antibodies that bind to specific signaling and co-stimulatory molecules.
Using flow cytometry, analyze fluorescence signals from BSLBs and T cells to quantify the synaptic output.
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.
繁體中文
