Primary murine peritoneal cells were sorted into B-1a and B-1b cells using MACS B-1a selection kit (Miltenyi Biotec Inc). Sorted cells were cultured in RPMI1640 containing 10% heat-inactivated fetal bovine serum (FBS, Wisent), 100 U/mL penicillin and streptomycin (Gibco), and 50 µM 2-mercaptoethanol (Amresco) for 3 days with 0.5 µg/mL LPS (Invivogen) and 0.5 µg/mL anti-IgM F(ab’)2 (Jackson ImmunoResearch). Cultures were collected, and cells were pelleted by centrifugation. Culture supernatants containing secreted antibodies were collected, and antibodies were purified by filtration using a 100 kDa centrifugation filter (retaining the > 100 kDa fraction). Antibody composition was determined by ELISA using mouse IgM/IgG quantitation set (Bethyl Laboratories), according to the manufacturer’s protocol. Total concentration of antibody was determined by BCA assay (Thermo Fisher Scientific).
Mouse igm igg quantitation set
Manufactured by Fortis Life Sciences
The Mouse IgM/IgG quantitation set is a laboratory tool designed for the quantitative measurement of mouse immunoglobulin M (IgM) and immunoglobulin G (IgG) levels in biological samples. This set provides the necessary reagents and protocols to perform these specific quantitative analyses.
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Lab products found in correlation
Penicillin and streptomycin, by Thermo Fisher Scientific (2 mentions)
Macs b 1a selection kit, by Miltenyi Biotec (2 mentions)
Bca assay, by Thermo Fisher Scientific (2 mentions)
Anti igm f ab 2, by Jackson ImmunoResearch (2 mentions)
2 mercaptoethanol, by Avantor (2 mentions)
Lipopolysaccharides (lps), by InvivoGen (2 mentions)
Fetal bovine serum (fbs), by Wisent (2 mentions)
2 protocols using mouse igm igg quantitation set
1
Enrichment and Characterization of Murine B-1 Cells
2
Murine B-1a and B-1b Cell Isolation and Culture
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Primary murine peritoneal cells were sorted into B-1a and B-1b cells using MACS B-1a selection kit (Miltenyi Biotec inc.). Sorted cells were cultured in RPMI1640 containing 10% heat-inactivated fetal bovine serum (FBS, Wisent), 100 U/mL penicillin and streptomycin (Gibco), and 50 µM 2mercaptoethanol (Amresco) for 3 days with 0.5 µg/mL LPS (Invivogen) and 0.5 µg/mL anti-IgM F(ab')2 (Jackson ImmunoResearch). Cultures were collected and cells were pelleted by centrifugation. Culture supernatants containing secreted antibodies were collected and antibodies were purified by filtration using a 100 kDa centrifugation filter (retaining the > 100 kDa fraction). Antibody composition was determined by ELISA using mouse IgM/IgG quantitation set (Bethyl Laboratories), according to the 29 manufacturer's protocol. Total concentration of antibody was determined by BCA assay (Thermo Fisher Scientific).
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