Protocol detail

Measuring Antigen Extraction and Trafficking in Cells

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To measure antigen extraction, PMSs containing either fluorescently labeled anti-human Igκ (GeneTex) or anti-rat Igκ (Thermo Fisher) were prepared as described (44 (link)). Cells were harvested and stained with near-IR Live/Dead marker (Thermo Fisher), anti-IgD Fab (SouthernBiotech), anti-CD10 (BD Biosciences), and the signal was quantified by flow cytometry (BD LSR II). To measure antigen trafficking, cells were placed on PMSs containing DyLight 550 and pHrodo avidin (Thermo Fisher) conjugated goat F(ab’)₂ anti-human Igκ (SouthernBiotech) and anti-human Igλ (SouthernBiotech) as described above. Cells were harvested, stained with near-IR Live/Dead marker (Thermo Fisher), anti-IgD Fab (SouthernBiotech), anti-CD10 (BD Biosciences), and the signal was quantified by flow cytometry (BD LSR II).

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Kwak K., Quizon N., Sohn H., Saniee A., Manzella-Lapeira J., Holla P., Brzostowski J., Lu J., Xie H., Xu C., Spillane K.M., Tolar P, & Pierce S.K. (2018). Intrinsic properties of human germinal-center B cells set antigen-affinity thresholds. Science immunology, 3(29), eaau6598.

Publication 2018

near-IR Live/Dead marker anti-IgD Fab anti-CD10 BD LSR II DyLight 550 anti-human Igλ

Anti igd Antigen Avidin Cells Flow cytometry Goat Human Pmss

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Variable analysis

independent variables

Fluorescently labeled anti-human Igκ (GeneTex)
Anti-rat Igκ (Thermo Fisher)

dependent variables

Antigen extraction
Antigen trafficking

control variables

Near-IR Live/Dead marker (Thermo Fisher)
Anti-IgD Fab (SouthernBiotech)
Anti-CD10 (BD Biosciences)
DyLight 550 and pHrodo avidin (Thermo Fisher) conjugated goat F(ab')2 anti-human Igκ (SouthernBiotech)
Anti-human Igλ (SouthernBiotech)

controls

Positive control: Not specified
Negative control: Not specified

Annotations

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