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2 protocols using 4 hydroxy 3 nitrophenylacetyl np ficoll

1

Cd244 Knockout Mice for Immunological Studies

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C57BL/6 (WT) mice were purchased from The Jackson Laboratories (Bar Harbor, ME).
Cd244-/- mice on the C57BL/6 background [12 (link)], were kindly provided by Dr. Raymond Welsh (University of Massachusetts Medical Center, Worcester, MA). Mice were maintained in the Translational Biomedical Research Center of the Medical College of Wisconsin and both genders were used for experiments in an age (6–12 week) and gender-matched manner. anti-CD45R-PE-Texas Red, anti-CD45R-PE, anti-GL7-FITC and anti-CD5-APC were purchased from BD Biosciences (San Jose, CA). CD21-eFluor 450, anti-CD23-PE-Cy7, anti-CD93-biotin, anti-CD4-APC-eFluor 780, anti-CD8-PE-Cy7, anti-TCRβ-FITC, anti-TCRβ-PE, anti-CD11b-Alexa Fluor 488, anti-CD244, anti-Foxp3-PE, anti-CD19-Alexa 700, anti-CD4-PE, anti-IgG-FITC and Streptavidin PE-Cy5.5 were purchased from eBioscience (San Diego, CA). Anti-CD11b-Brilliant Violet 605, anti-CD11c-PE, anti-NK1.1-APC, anti-IgM-APC Cy7, anti-IgD-Pacific Blue, anti-CD38-Alexa Fluor 647, anti-CD138-APC and rat anti-mouse IgG2a-biotin were purchased from Biolegend (San Diego, CA). Anti-IgM-FITC and the SBA Clonotyping System-B6/C57J-HRP were purchased from Southern Biotech (Birmingham, AL). 4-Hydroxy-3-nitrophenylacetyl (NP)-ficoll, NP-Chicken Gamma Globulin (CGG), NP(24)-PE and NP-BSA were purchased from Biosearch Technologies (Novato, CA).
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2

Antigen-specific B cell response

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Eight to ten week old BALB/c and Caspase 1−/− mice were vaccinated intraperitoneally with an emulsion of 50% V/V Incomplete Freund’s adjuvant (Sigma-Aldrich) and either: 100ul PBS, 100ug 4-Hydroxy-3-nitrophenylacetyl (NP)-Ficoll (Biosearch Technologies), or 100ug NP-Ovalbumin (Biosearch Technologies). Serum was collected by saphenous vein bleed at weekly intervals.
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