Longitudinal sampling of fecal pellets from experimental mice was performed to quantify the dynamics of IgA responses across adoptive transfer treatment groups. To do this, fecal pellets were collected immediately prior to adoptive transfer and at weekly intervals thereafter until 7 weeks post-transfer. Fecal pellets were crushed in 500μL of Hank’s Balanced Salt Solution (HBSS) and centrifuged at 4000xG for 10 minutes. Subsequently, the supernatant was transferred to new tubes and centrifuged at 8000xG for 10 minutes. The remaining supernatant was collected. These samples were subsequently measured for IgA or IgM content by ELISA, using the Invitrogen IgA Mouse Uncoated ELISA kit (catalog #88-50450-88) or the Invitrogen IgM Mouse Uncoated ELISA kit (catalog #88-50470-22). The IgA/IgM content of these samples was determined, and the values were standardized by weight of pellet.
Igm mouse uncoated elisa kit
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
The IgM Mouse Uncoated ELISA Kit is a laboratory instrument used to detect and measure the levels of mouse immunoglobulin M (IgM) in biological samples. It is designed to provide quantitative results through an enzyme-linked immunosorbent assay (ELISA) technique.
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Lab products found in correlation
Iga mouse uncoated elisa kit, by Thermo Fisher Scientific (2 mentions)
Anti cd40, by BioXCell (2 mentions)
Anti igm f ab 2, by BioXCell (2 mentions)
Goat igg2a, by BioXCell (2 mentions)
B cell magnisort kit, by Thermo Fisher Scientific (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Ig isotyping mouse uncoated elisa kit, by Thermo Fisher Scientific (1 mentions)
4 protocols using igm mouse uncoated elisa kit
1
Longitudinal Fecal IgA Quantification
2
SRBC-Induced B Cell Activation Modulation
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10d after immunizing groups of control Cγ1-Cre+/−, DAF-TMCγ1 and ΔC3ar1/C5ar1Cγ1 mice with SRBC, animals were injected with anti-CD40 (150ug) and/or anti-IgM F(ab’)2 (100ug), or goat IgG2a (all from BioXcell) in 200ul PBS i.v. Spleens were harvested at 4h for surface staining followed by fixation, permeabilization and intracellular staining. In other studies, 20×106 B6 WT or C3aR1−/−C5aR1−/− B cells were transferred into μMT recipients and 24 h later immunized the recipients with SRBC. On d10, animals were injected with anti-CD40 (150ug) and/or anti-IgM F(ab’)2 (100ug), or control goat IgG2a and the cells analyzed by flow cytometry. Serum IgM was quantified with an IgM Mouse Uncoated ELISA Kit (Invitrogen).
For in vitro studies, 10d after sequential SRBC immunization, isolated spleen cells were enriched for B cells (B cell Magnisort kit, ThermoFisher), rested for 20min at 37C, 5% CO2 in HL-media and subsequently stimulated with 10ug/ml anti-CD40, 10ug/ml anti-IgM, 0.5ug/ml murine C3a and or C5a for 20’ at 37C, 5% CO2. After washing, cells were stained and analyzed cells by flow cytometry.
For in vitro studies, 10d after sequential SRBC immunization, isolated spleen cells were enriched for B cells (B cell Magnisort kit, ThermoFisher), rested for 20min at 37C, 5% CO2 in HL-media and subsequently stimulated with 10ug/ml anti-CD40, 10ug/ml anti-IgM, 0.5ug/ml murine C3a and or C5a for 20’ at 37C, 5% CO2. After washing, cells were stained and analyzed cells by flow cytometry.
3
Quantification of Immunoglobulins in Tissues and Serum
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For quantification of immunoglobulins, tissues were dissected and homogenized in ice-cold RIPA buffer (Thermo Fisher Scientific, UK). After 20 min on ice, the lysates were centrifuged for 10 min at 10,000 g, and the clear supernatant was transferred to a new Eppendorf tube. For serum samples, blood was withdrawn by cardiac puncture, incubated for 30 min at room temperature, and centrifuged for 10 min at 2,000 g at 4°C. The clear supernatant was transferred to a new tube. All samples were stored at −80°C until further use. Immunoglobulin A (IgA) was measured with the IgA Mouse Uncoated ELISA Kit, Immunoglobulin M (IgM) was measured with the IgM Mouse Uncoated ELISA Kit, and all the immunoglobulins were measured with an Ig Isotyping Mouse Uncoated ELISA Kit (all from Invitrogen, Thermo Fisher Scientific, UK).
4
SRBC-Induced B Cell Activation Modulation
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