Briefly, Immulon 4 HBX 96-well microtiter plates (VWR International, Randor PA) were coated with mouse anti-human IgM antibody (Sigma Aldrich) at a concentration of 1 μg/mL overnight. Culture media collected from activated B cell cultures were collected and incubated on antibody coated plates for 2 h at 37°C with 5% CO2. This was followed by a 1 h incubation at 37°C with a horseradish peroxidase-conjugated anti-IgM secondary antibody (Sigma Aldrich). Culture plates were washed 3 times with PBS containing 0.05% Tween-20 (Sigma Aldrich) and 3 times with nanopure water after each incubation. 2,2’-Azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS, Sigma Aldrich) was used as a colorimetric substrate for HRP. Kinetic readings of 405 nm for 1 h with a Synergy HT microplate reader (Biotek, Winooski, VT) were performed to determine OD. IgM concentration in culture supernatants was calculated based on a standard curve and normalized to viable cell number.
Immulon 4 hbx 96 well microtiter plates
Manufactured by Avantor
Immulon 4 HBX 96-well microtiter plates are a laboratory product designed for use in various immunological assays. These plates feature a high-binding surface to facilitate the immobilization of proteins, peptides, or other biomolecules. The plates are made of polystyrene material and are available in a standard 96-well format.
Automatically generated - may contain errors
Lab products found in correlation
Tween 20, by Merck Group (3 mentions)
Synergy ht microplate reader, by Agilent Technologies (2 mentions)
2 2 azinobis 3 ethylbenzothiazoline 6 sulfonic acid (abts), by Merck Group (2 mentions)
2 2 azino bis, by Merck Group (2 mentions)
Mouse anti human igm antibody, by Merck Group (1 mentions)
Anti human igm antibody, by Merck Group (1 mentions)
Synergy ht, by Agilent Technologies (1 mentions)
2 2 azinobis, by Roche (1 mentions)
3 protocols using immulon 4 hbx 96 well microtiter plates
1
ELISA for Quantifying IgM Secretion
2
Quantification of IgM Antibodies by ELISA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The quantity of IgM antibodies secreted into the culture supernatants was determined by sandwich ELISA. Briefly, Immulon 4 HBX 96-well microtiter plates (VWR International, Radnor, Pennsylvania) were coated with anti-human IgM antibody (1 μg/ml; Sigma Aldrich, St. Louis, MO) overnight. Supernatants collected from human B cell cultures were incubated over primary antibody-coated plates for 90 min at 37°C with 5% CO2 and followed by overlaying an anti-human IgM-HRP conjugate antibody (Sigma Aldrich, St. Louis, MO). Each of the incubations was followed by washes with phosphate-buffered saline (pH 7.4) containing 0.05% Tween-20 (Sigma Aldrich, St. Louis, MO) and nanopure water. 2,2’-Azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS, Roche Diagnostics) was then added as a colorimetric substrate for HRP. The rate of colorimetric change was quantified with a Synergy HT microplate reader (BioTek, Winooski, Vermont) at 405 nm for 1 h. The concentration of IgM in supernatants was calculated based on a standard curve created in each plate.
3
Quantification of IgM and IgG Secretion
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The concentration of IgM and IgG secreted into the culture supernatant was quantified by sandwich ELISA. In brief, Immulon 4 HBX 96-well microtiter plates (VWR International, Radnor, Pennsylvania) were coated with anti-human IgM and IgG antibodies (both 1 μg/ml; Sigma-Aldrich) overnight. Culture media collected from human B cells were incubated over primary antibody-coated plates for 90 min at 37°C with 5% CO2 and was followed by overlaying with anti-human IgM-HRP or anti-human IgG-HRP conjugated antibodies (Sigma-Aldrich). Incubations were followed by washes with phosphate-buffered saline (pH 7.4) containing 0.05% Tween-20 (Sigma-Aldrich) and PBS. 2,2’-Azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS, Sigma-Aldrich) was then added as a colorimetric substrate for HRP. The rate of colorimetric change was quantified with a Synergy HT microplate reader (BioTek, Winooski, Vermont) at 405 nm for 1 h. Concentrations of IgM and IgG in media were calculated based on a standard curve created in each plate with purified human IgM and IgG standards (Sigma Aldrich).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!