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C57bl 6 mouse immunoglobulin panel

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The C57BL/6 Mouse Immunoglobulin Panel is a laboratory reagent used to detect and quantify immunoglobulins in C57BL/6 mouse samples. It includes a set of antibodies specific to different mouse immunoglobulin isotypes.

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Lab products found in correlation

Tmb solution, by Thermo Fisher Scientific (5 mentions) Goat anti mouse ig, by Southern Biotech (4 mentions) Hopc 1, by Southern Biotech (2 mentions) Duoset elisa kit, by R&D Systems (2 mentions) Mouse il 13 duoset elisa, by R&D Systems (1 mentions) αgm csf, by BioLegend (1 mentions) Opteia 555214, by BD (1 mentions) Murine gm csf, by Thermo Fisher Scientific (1 mentions)

7 protocols using c57bl 6 mouse immunoglobulin panel

1

Cytokine and Antibody ELISA Assays for Helminth Infection

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Nunc Maxisorp plates were coated with capture antibodies (for cytokine ELISAs) or H. polygyrus lysate (for H. polygyrus-specific IgG1 ELISA) overnight. Following blocking, culture supernatants (cytokine ELISAs) or serum (IgG1 ELISA) were added, followed by incubation with cytokine-detection biotinylated antibodies or horseradish peroxidase (HRP)-conjugated anti-mouse IgG1 antibody. For cytokine ELISAs, streptavidin-conjugated horseradish peroxidase (HRP) was added. Substrate solution (BD OptEIA 555214) was added, reactions were stopped with 1 M H3PO4, and absorbance was read at OD450. For cytokine ELISAs, standard curves were generated with purified cytokines. For the IgG1 ELISA, serum was diluted between 10−2 and 10−8 and the last well with an OD450 above 0.100 represented the titer. The Mouse IL-4 and IL-5 ELISA Max Standard Sets (BioLegend), Mouse IL-13 DuoSet ELISA (R&D), and C57BL/6 Mouse Immunoglobulin Panel (SouthernBiotech) were used. For detection of GM-CSF, biotinylated αGM-CSF (BioLegend, MP1–31G6), unconjugated αGM-CSF (BioLegend, MP1–22E9), and murine GM-CSF (Peprotech) were used.
To generate H. polygyrus lysate, adult worms were washed in PBS and ground in 1 mL of PBS. Debris was pelleted by centrifugation at 16,000 g for 20 minutes at 4 °C. The supernatant was passed through a 0.2 μm filter and stored at −80 °C.
Jarjour N.N., Bradstreet T.R., Schwarzkopf E.A., Cook M.E., Lai C.W., Huang S.C., Taneja R., Stappenbeck T.S., Van Dyken S.J., Urban JF J.r, & Edelson B.T. (2020). BHLHE40 promotes TH2 cell-mediated anti-helminth immunity and reveals cooperative CSF2RB family cytokines. Journal of immunology (Baltimore, Md. : 1950), 204(4), 923-932.
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2

Murine Serum Immunoglobulin Profiling

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Blood was collected, as previously described, and analyzed for serum immunoglobulins (IgM, IgG1, IgG2c, IgG2b, IgG3, IgA and IgE) using the C57BL/6 Mouse Immunoglobulin Panel (Southern Biotech), following manufacturer’s instructions. Serum levels of anti-nuclear antigens were determined using the Mouse Anti-Nuclear Antigens (ANA/ENA) Ig’s (total (A+G+M)) ELISA Kit (Alpha Diagnostics International), following manufacturer’s instructions.
O’Hagan K.L., Miller S.D, & Phee H. (2017). Pak2 is essential for the function of Foxp3+ regulatory T cells through maintaining a suppressive Treg phenotype. Scientific Reports, 7, 17097.
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3

ELISA for Virus-specific Antibodies

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Ninety six-well EIA/RIA plate wells were filled with 100 µl of heat-inactivated purified HSV-2 (104 to 105 pfu equivalent per 100 µl) or heat-inactivated purified VSV (5 × 105 pfu equivalent per 100 µl) for virus-specific Ig measurement or goat anti-mouse Ig (1:1000; SouthernBiotech, 1010-01) for total Ig measurement in carbonate buffer (pH 9.5) and then incubated overnight at 4°C. On the following day, these plates were washed with PBS-Tween 20 and blocked 2hr with 5% FBS in PBS. Tissue samples and serum samples in ABC buffer were then plated in the wells and incubate for at least four hours at ambient temperature. After washing in PBS-Tween 20, HRP-conjugated anti-mouse IgG1, IgG3, IgM, IgA, IgG2a, IgG2b or IgG2c (SouthernBiotech) was added in the wells for 1 h, followed by washing and adding TMB solution (eBioscience). Reactions were stopped with 1N H2SO4 and absorbance was measured at 450 nm. The sample Ab titers were defined by using Ig standard (C57BL/6 Mouse Immunoglobulin Panel; SouthernBiotech) or mouse IgG2a (HOPC-1; SouthernBiotech).
Iijima N, & Iwasaki A. (2016). Access of protective antiviral antibody to neuronal tissues requires CD4 T cell help. Nature, 533(7604), 552-556.
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4

Measurement of Virus-Specific Antibody Responses

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Vaginal fluids were collected from the indicated days after infection by using calcium alginate swabs and pipetting PBS
into and out of the vagina 20 times. BAL were collected by washing the trachea with 1 mL of PBS containing 0.1% BSA. Ninety
six-well EIA/RIA plate was coated with 100 µl of heat-inactivated or PFA-inactivated purified HSV-2 (104 to
105 pfu equivalent per 100 µl) or PFA-inactivated A/PR8 virion (105 pfu equivalent per 100
µl) for virus-specific Ig measurement or goat anti-mouse Ig (1:1000; SouthernBiotech, 1010–01) for total Ig
measurement in carbonate buffer (pH 9.5) and then incubated overnight at 4°C. On the following day, these plates were
washed with PBS-Tween 20 and blocked 2hr with 5% FBS in PBS. Wash and serum samples were then plated in the wells and incubate for
at least four hours at ambient temperature. After washing in PBS-Tween 20, HRP-conjugated anti-mouse IgG1, IgG3, IgM, IgA, IgG2a,
IgG2b or IgG2c (SouthernBiotech) was added in the wells for 1 h, followed by washing and adding TMB solution (eBioscience).
Reactions were stopped with 1N H2SO4 and absorbance was measured at 450 nm. The total Ab titers were defined
by using Ig standard (C57BL/6 Mouse Immunoglobulin Panel; SouthernBiotech). The levels of CXCL9 and CXCL10 in vaginal fluids were
measured using DuoSet ELISA kit (R&D Systems) according to manufacturer’s instructions.
Oh J.E., Iijima N., Song E., Lu P., Klein J., Jiang R., Kleinstein S.H, & Iwasaki A. (2019). Migrant memory B cells secrete luminal antibody in the vagina. Nature, 571(7763), 122-126.
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5

Measurement of Virus-Specific Antibody Responses

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Vaginal fluids were collected from the indicated days after infection by using calcium alginate swabs and pipetting PBS
into and out of the vagina 20 times. BAL were collected by washing the trachea with 1 mL of PBS containing 0.1% BSA. Ninety
six-well EIA/RIA plate was coated with 100 µl of heat-inactivated or PFA-inactivated purified HSV-2 (104 to
105 pfu equivalent per 100 µl) or PFA-inactivated A/PR8 virion (105 pfu equivalent per 100
µl) for virus-specific Ig measurement or goat anti-mouse Ig (1:1000; SouthernBiotech, 1010–01) for total Ig
measurement in carbonate buffer (pH 9.5) and then incubated overnight at 4°C. On the following day, these plates were
washed with PBS-Tween 20 and blocked 2hr with 5% FBS in PBS. Wash and serum samples were then plated in the wells and incubate for
at least four hours at ambient temperature. After washing in PBS-Tween 20, HRP-conjugated anti-mouse IgG1, IgG3, IgM, IgA, IgG2a,
IgG2b or IgG2c (SouthernBiotech) was added in the wells for 1 h, followed by washing and adding TMB solution (eBioscience).
Reactions were stopped with 1N H2SO4 and absorbance was measured at 450 nm. The total Ab titers were defined
by using Ig standard (C57BL/6 Mouse Immunoglobulin Panel; SouthernBiotech). The levels of CXCL9 and CXCL10 in vaginal fluids were
measured using DuoSet ELISA kit (R&D Systems) according to manufacturer’s instructions.
Oh J.E., Iijima N., Song E., Lu P., Klein J., Jiang R., Kleinstein S.H, & Iwasaki A. (2019). Migrant memory B cells secrete luminal antibody in the vagina. Nature, 571(7763), 122-126.
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6

Quantifying HSV-2-Specific Antibodies in Mice

Cited 1 time
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CSF and serum were collected from mice as previously described9 (link). The serum and CSF were then diluted with PBS containing 0.1% BSA in a 1:1 ratio. Plates (96-well) were coated with 100 μl of heat-inactivated or PFA-inactivated purified HSV-2 (104 to 105 plaque-forming units equivalent per 100 μl) for virus-specific immunoglobulin measurement or goat anti-mouse immunoglobulin (SouthernBiotech, 1010-01, 1:1,000) and then incubated overnight at 4 °C. These plates were then washed with PBS–Tween 20 and blocked for 2 h with 5% FBS in PBS. Samples were then plated in the wells and incubated for at least 4 h at room temperature. After being washed in PBS–Tween 20, HRP-conjugated anti-mouse immunoglobulin antibodies (SouthernBiotech, 1010-05, 1:5,000) were added in the wells for 1 h, followed by washing and addition of TMB solution (eBioscience). Reactions were stopped with 1 N H2SO4, and absorbance was measured at 450 nm. The total antibody titres were defined by using an immunoglobulin standard (C57BL/6 mouse immunoglobulin panel; SouthernBiotech).
Yin X., Zhang S., Lee J.H., Dong H., Mourgkos G., Terwilliger G., Kraus A., Geraldo L.H., Poulet M., Fischer S., Zhou T., Mohammed F.S., Zhou J., Wang Y., Malloy S., Rohner N., Sharma L., Salinas I., Eichmann A., Thomas J.L., Saltzman W.M., Huttner A., Zeiss C., Ring A., Iwasaki A, & Song E. (2024). Compartmentalized ocular lymphatic system mediates eye–brain immunity. Nature, 628(8006), 204-211.
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7

ELISA for Virus-specific Antibodies

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Ninety six-well EIA/RIA plate wells were filled with 100 µl of heat-inactivated purified HSV-2 (104 to 105 pfu equivalent per 100 µl) or heat-inactivated purified VSV (5 × 105 pfu equivalent per 100 µl) for virus-specific Ig measurement or goat anti-mouse Ig (1:1000; SouthernBiotech, 1010-01) for total Ig measurement in carbonate buffer (pH 9.5) and then incubated overnight at 4°C. On the following day, these plates were washed with PBS-Tween 20 and blocked 2hr with 5% FBS in PBS. Tissue samples and serum samples in ABC buffer were then plated in the wells and incubate for at least four hours at ambient temperature. After washing in PBS-Tween 20, HRP-conjugated anti-mouse IgG1, IgG3, IgM, IgA, IgG2a, IgG2b or IgG2c (SouthernBiotech) was added in the wells for 1 h, followed by washing and adding TMB solution (eBioscience). Reactions were stopped with 1N H2SO4 and absorbance was measured at 450 nm. The sample Ab titers were defined by using Ig standard (C57BL/6 Mouse Immunoglobulin Panel; SouthernBiotech) or mouse IgG2a (HOPC-1; SouthernBiotech).
Iijima N, & Iwasaki A. (2016). Access of protective antiviral antibody to neuronal tissues requires CD4 T cell help. Nature, 533(7604), 552-556.
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