Antibodies used for flow cytometry analysis were: anti-human/mouse CD45 (Invitrogen, # 17-0451-82), anti-human CD20 (Invitrogen, #25-0209-42), anti-human MARCH3 (Invitrogen, #PA5-60351), biotin-anti-B2M (Abcam, #ab269365), anti-human DTWD1 (Invitrogen, #MA5-27492), anti-human IgG1 (Abcam, #ab99776), anti-human RPS12 (Abcam, #ab175219) and anti-human IgG4 (Abcam, #ab1930).
The detailed staining procedure of flow cytometry analysis has been described previously (13 (link)). Briefly, nonspecific binding was blocked with Fcγ receptor blocker (Invitrogen, #14-9161-73). For surface staining, cells were incubated on ice with fluorescent-coupled antibodies for 15 min and then washed twice with cold PBS. For intracellular staining, cells were treated using a Cytofix/Cytoperm kit (BD, #554717) according to manufacturer’s introductions and then incubated at 4°C with the relevant fluorescent-coupled antibodies for 15 min. After two washes with cold PBS, cells were re-suspended with PBS/0.5%BSA/2mM EDTA. Samples were then measured with a flow cytometer (Thermal Fisher Attune NxT).
The detailed staining procedure of flow cytometry analysis has been described previously (13 (link)). Briefly, nonspecific binding was blocked with Fcγ receptor blocker (Invitrogen, #14-9161-73). For surface staining, cells were incubated on ice with fluorescent-coupled antibodies for 15 min and then washed twice with cold PBS. For intracellular staining, cells were treated using a Cytofix/Cytoperm kit (BD, #554717) according to manufacturer’s introductions and then incubated at 4°C with the relevant fluorescent-coupled antibodies for 15 min. After two washes with cold PBS, cells were re-suspended with PBS/0.5%BSA/2mM EDTA. Samples were then measured with a flow cytometer (Thermal Fisher Attune NxT).
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