Fresh PBMCs were cultured for 6 days in the presence of a nonspecific B-cell stimulant containing 83 ng/mL of pokeweed mitogen extract (Sigma-Aldrich), 2.5 μg/mL of fully methylated CpG (Invitrogen), and a 1:5,000 dilution of fixed Staphylococcusaureus, Cowan (SAC, Calbiochem), which stimulates polyclonal B memory cells to proliferate and differentiate into antibody-secreting cells. After culture, supernatants were preserved at −80 °C for further cytokine analysis (Cytokine immunoassays, see Methods, Supplementary Digital Content 1, http://links.lww.com/CTG/A16 ).
Pokeweed mitogen extract
Manufactured by Merck Group
Pokeweed mitogen extract is a laboratory reagent derived from the pokeweed plant. It functions as a mitogen, a substance that stimulates cell division and proliferation in lymphocytes, which are a type of white blood cell. The extract is commonly used in cell biology research and assays to activate and study the immune response in vitro.
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Lab products found in correlation
3 protocols using pokeweed mitogen extract
1
Polyclonal B-cell Stimulation and Cytokine Analysis
2
Detecting Antigen-specific Memory B Cells
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Antigen-specific memory B cells (MBC) were detected essentially as previously described [30 (link)]. Briefly, PBMC were cultured at 1x106 cells per ml of R10 supplemented with 50 uM B-mercaptoethanol (Sigma-Aldrich) and polyclonally stimulated with pokeweed mitogen extract (1 μg/mL, Sigma-Aldrich), phosphothiolated CpG ODN-2006 (6 μg/mL, Invivogen), and Staphylococcus aureus Cowan (1:10,000, Sigma-Aldrich) for 6 days. After in vitro stimulation, total and V. cholerae specific IgM, IgG, and IgA cells were quantified by ELISPOT assay, as described above.
3
Enumeration of Dengue-Specific Memory B Cells
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The frequency of dengue specific MBCs were enumerated (n = 56) by 6 days polyclonal stimulation using standard protocol (Crotty et al., 2004) . PBMCs were washed with RPMI-1640 containing 10% FCS and cultured in 24 well plate at 5X10 5 cells/ well in RPMI supplemented with stimulation mix 1:100,000 pokeweed mitogen extract (Sigma; L9379), 6 mg/ml fully phosphothioated CpG ODN-2006 (Qiagen) , and 1/10,000 Staphylococcus Aureus Cowan (Sigma; P7155) for 6 days. Cultured cells were then thoroughly washed and plated in to ELISPOT plates precoated with anti-human Ig or dengue antigen. Plates were then incubated overnight at 37 C with 5% CO 2 . Next day cells were removed and the IgG production was assessed by incubation with secondary biotinylated antibody to human Ig and further washed six times. The plates were developed with Avidin-HRP (Mabtech AB) and 3-amino-9-ethylcarbazole (AEC) as substrate, and the spots enumerated using an automated ELISPOT reader. Frequency of dengue specific MBCs were calculated as a fraction of total IgG spots.
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