Tracking Antigen-Specific B and T Cells

Kaede OT2 T cells were enriched by negative depletion with biotinylated antibodies for anti-B220 clone RA3-6B2, anti-CD11b clone M1/70, anti-CD11c clone HL3, anti-CD8 clone 53–6.7, and tdtomato SW_HEL B cells (Phan et al., 2003 (link)) were enriched by negative depletion with biotinylated antibodies for anti-CD11b, anti-CD11c, anti-CD4 clone GK1.5, anti-CD43 clone S7 (all from BD Bisociences) and MACs anti-biotin magnetic beads (Miltenyi). 2.5 × 10⁵ B220⁺ HEL⁺ SWHEL tdTomato B cells and V_α2⁺ CD4⁺ Kaede OT2 cells were adoptively transferred into C57BL/6 recipients and immunised the next day with 20 μg HEL-OVA in Sigma Adjuvant System (Sigma) injected subcutaneously in the lower flank and tail base. To label SCS macrophages, we injected CD169 clone Ser-4 (UCSF Hybridoma Core) conjugated to Alexa Fluor 680 (Invitrogen), 12 hr before imaging. Mice were imaged 7 days after immunisation.

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Warren S.C., Nobis M., Magenau A., Mohammed Y.H., Herrmann D., Moran I., Vennin C., Conway J.R., Mélénec P., Cox T.R., Wang Y., Morton J.P., Welch H.C., Strathdee D., Anderson K.I., Phan T.G., Roberts M.S, & Timpson P. (2018). Removing physiological motion from intravital and clinical functional imaging data. eLife, 7, e35800.

Publication 2018

MACs anti-biotin magnetic beads Sigma Adjuvant System Alexa Fluor 680

Adjuvant Anti antibodies B cells Biotin Cd11b Cd4 cells Cd43 Clone Hybridoma Immunisation Macrophages Mice T cells Tail Tdtomato

Corresponding Organization :

Other organizations : UNSW Sydney, Garvan Institute of Medical Research, St Vincent's Clinic, University of Queensland, Therapeutics Clinical Research, La Jolla Bioengineering Institute, University of California, San Diego, Cancer Research UK Scotland Institute, Babraham Institute, The Francis Crick Institute, University of South Australia

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Variable analysis

independent variables

Enrichment of Kaede OT2 T cells by negative depletion with biotinylated antibodies for anti-B220, anti-CD11b, anti-CD11c, and anti-CD8
Enrichment of tdtomato SW_HEL B cells by negative depletion with biotinylated antibodies for anti-CD11b, anti-CD11c, anti-CD4, and anti-CD43
Adoptive transfer of 2.5 × 10⁵ B220⁺ HEL⁺ SWHEL tdTomato B cells and V_α2⁺ CD4⁺ Kaede OT2 cells into C57BL/6 recipients
Immunization with 20 μg HEL-OVA in Sigma Adjuvant System injected subcutaneously in the lower flank and tail base
Labeling of SCS macrophages with CD169 clone Ser-4 conjugated to Alexa Fluor 680, 12 hr before imaging

dependent variables

Observations and measurements made during the imaging of mice 7 days after immunization

control variables

C57BL/6 recipients used for adoptive transfer

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

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