Enumeration of 4-44+ and NP-specific AFCs

Detection of 4-44⁺ AFCs (35 (link)) and NP-specific AFCs using ELISpot has been described (50 (link)). Briefly, Immulon 4 plates were coated with either anti-mouse IgM (B7-6, produced in lab) or polyclonal anti-mouse IgG2a (Southern Biotech) for 4-44 AFCs or with NP₂-BSA or NP₁₆-BSA and blocked with PBS and 1% BSA. NP-BSA conjugates were produced and characterized as described (49 (link)). Splenocytes were incubated at 37 degrees with 5% CO₂ for 4.5-6 hours. 4-44⁺ AFCs were enumerated by detection with 4-44 biotin and Streptavidin-alkaline phosphatase while NP AFCs were detected using anti-IgM or anti-IgG1 alkaline phosphatase (Southern Biotech). Color was developed using 5-Bromo-4-chloro-3-indolyl phosphate (BCIP) in agarose. AFCs were counted using a dissecting microscope.

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Sweet R.A., Nickerson K.M., Cullen J.L., Wang Y, & Shlomchik M.J. (2017). B Cell Extrinsic Myd88 and Fcer1g Negatively Regulate Autoreactive and Normal B cell Immune Responses. Journal of immunology (Baltimore, Md. : 1950), 199(3), 885-893.

Publication 2017

anti-mouse IgG2a

5 bromo 4 chloro 3 indolyl phosphate Agarose Alkaline phosphatase Anti igm Biotin Elispot Igg1 Igg2a Microscope Mouse Streptavidin

Corresponding Organization : University of Pittsburgh

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Variable analysis

independent variables

Coating of Immulon 4 plates with either anti-mouse IgM (B7-6) or polyclonal anti-mouse IgG2a for 4-44 AFCs, or with NP2-BSA or NP16-BSA for NP-specific AFCs

dependent variables

Enumeration of 4-44+ AFCs by detection with 4-44 biotin and Streptavidin-alkaline phosphatase
Enumeration of NP-specific AFCs using anti-IgM or anti-IgG1 alkaline phosphatase

control variables

Incubation of splenocytes at 37 degrees with 5% CO2 for 4.5-6 hours
Blocking of Immulon 4 plates with PBS and 1% BSA

positive controls

NP2-BSA or NP16-BSA conjugates for NP-specific AFCs

negative controls

Not explicitly mentioned

Annotations

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