For all imaging experiments, glass-bottom chambers (World Precision Instruments, FD3510) were used for coating, whereas Western blotting experiments were performed in coated plastic 24-well tissue culture plates. For TNPBSA-IgE coating, surfaces were incubated with 200 μl of TNP-conjugated BSA (TNPBSA; 50 μg/ml) (Insight Biotechnology) in coating buffer [50 mM Na2CO3 and 50 mM NaHCO3 (pH 9.6)] overnight at 4°C. The chambers were washed three times with 200 μl of PBS before 200 μl of mouse anti-TNP IgE (5 μg/ml, unless otherwise specified; BD Biosciences, catalog no. 554118) in PBS was added to the chamber and incubated at 4°C for 2 hours. Chambers were then washed three times with 200 μl of PBS and covered with 200 μl for PBS for experiments. For PLL coating, surfaces were incubated with 200 μl of 0.01% PLL for 30 min at room temperature and then washed three times with 200 μl of PBS before use. For laminin peptide coating, surfaces were coated with 200 μl of rat laminin peptide (150 μg/ml; Merck Millipore, catalog no. SCR127) and TNPBSA in coating buffer (50 μg/ml) overnight at 4°C. Chambers were washed and coated with IgE as described earlier. For Der f 1 coating, surfaces were coated with 200 μl of purified Der f 1 (50 μg/ml; Indoor Biotechnologies, catalog no. NA-DF1-2) in coating buffer overnight at 4°C and washed three times with 200 μl of PBS before use. Before they were stimulated on these surfaces, primary basophils were loaded with human anti–Der f 1 IgE (1 μg/ml; Absolute Antibody, Ab00132-14.0) for 10 min and then washed three times with PBS. For anti-IgE coating, surfaces were coated with either 200 μl of mouse anti-hIgE (50 μg/ml; BioLegend, catalog no. 325507) or donkey anti–mouse IgG (50 μg/ml; Stratech, 715-001-003 JIR) in coating buffer overnight at 4°C. Surfaces were washed three times with 200 μl of PBS, and then, the donkey anti-mouse–coated surfaces were incubated with mouse anti-hIgE (5 μg/ml) for 2 hours at room temperature before washing three times with 200 μl of PBS. Coating efficiency was determined with anti-hIgE labeled with the Alexa Fluor 647 antibody labeling kit (Thermo Fisher Scientific) as per the manufacturer’s instructions, with fluorescence intensity at the coverslip surface measured with a Zeiss LSM 780. SLBs were prepared as described previously (37). Briefly, glass slides were cleaned with piranha solution. Lipid mix [1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC):nickelated lipid, 96:4; 1 mg/ml] was deposited on the glass slides on a spin coater at 3000 rpm for 30 s and hydrated in Hepes-buffered saline (HBS). His-Fcε (1 μg/ml) or equimolar equivalents of other Fcε chimeras were coated and washed as described previously (37). Ni-coated glass coverslips (Nanocs, catalog no. CS-NTA-5) were coated with His-Fcε (1 μg/ml) or equimolar equivalents of other Fcε chimeras for 30 min before being washed three times with 500 μl of PBS. Coverslips were then blocked with 5% BSA for 30 min and washed three times with 500 μl of PBS.

Note: The content above has been extracted from a research article, so it may not display correctly.



Q&A
Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.



We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.