Differentiation of MBCs into ASCs in was initiated in bulk PBMC cultures based on a previously established protocol [5] (link), using a stimulation cocktail composed of pokeweed mitogen (PWM), S. aureus Cowan I protein A (SAC) and CpG. IL-10 was added to the stimulation mix since a previous study showed that this enhanced the efficiency of MBC into ASC differentiation by more than 9 fold [6] (link). Briefly, PBMCs were thawed for 30 sec in a 37°C water bath and cold RPMI medium was immediately added drop wise. After washing, the cells were re-suspended in RPMI containing 10% FCS, 100 U/ml penicillin/streptomycin, 100 mM HEPES, 50 mM 2-β-Mercaptoethanol and 2 mM L-Glutamine (all Invitrogen) and counted. 1×106 cells/ml were added to 25 cm2 cell culture flasks (Greiner). Culture medium was supplemented with 50 ng/ml PWM lectin derived from Phytolacca americana (Sigma-Aldrich), 1∶5000 Protein A from Staphylococcus aureus, Cowan Strain (Sigma-Aldrich), 2.5 µg/ml ODN 2006 (Type B CpG nucleotide-human TLR9 ligand; InvivoGen tlrl-2006) and 25 ng/ml recombinant human IL-10 (PeproTech) and incubated at 37°C, 5% CO2 for 5 days.
Cowan strain
Manufactured by Merck Group
The Cowan Strain is a laboratory equipment product. It serves as a culture medium for the growth and maintenance of bacterial strains. The Cowan Strain provides a standardized and consistent substrate for cultivating specific microorganisms in a controlled laboratory setting.
Automatically generated - may contain errors
Lab products found in correlation
Streptavidin alkaline phosphatase, by Southern Biotech (2 mentions)
Immunocapture 6, by Cellular Technology (2 mentions)
Lectin pokeweed mitogen, by Merck Group (2 mentions)
Anti iga biotin, by Mabtech (2 mentions)
Nbt bcip, by Thermo Fisher Scientific (2 mentions)
25 cm2 cell culture flask, by Greiner (1 mentions)
Odn2006, by InvivoGen (1 mentions)
2 β mercaptoethanol, by Thermo Fisher Scientific (1 mentions)
Hepes, by Thermo Fisher Scientific (1 mentions)
Recombinant human il 10, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
3 protocols using cowan strain
1
MBC Differentiation into ASCs
2
SARS-CoV-2 Spike Protein ELISpot Assay
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MBC stimulations were performed on peripheral blood mononuclear cells (PBMCs) collected from subjects in the convalescent cohort. To induce MBC differentiation into antibody secreting cells, 1×106 PBMCs were stimulated with 10 ng/ml Lectin Pokeweed Mitogen (Sigma-Aldrich), 1/100,000 Protein A from Staphylococcus aureus, Cowan Strain (Sigma-Aldrich), and 6 μg/ml CpG (Invitrogen) in complete RPMI in an incubator at 37°C/5% CO2 for 5 days. After stimulation, cells were counted and added to ELISpot white polysterene plates (Thermo Fisher) coated with 4 μg/ml of SARS-CoV-2 spike that were blocked with 200 μl of complete RPMI. ELISpot plates were incubated with cells for 16 hours overnight in an incubator at 37μC/5% CO2. After the overnight incubation, plates were washed and incubated with anti-IgG-biotin and/or anti-IgA-biotin (Mabtech) for 2 hours at room temperature. After secondary antibody incubation, plates were washed and incubated with streptavidin-alkaline phosphatase (Southern Biotech) for 2 hours at room temperature. Plates were washed and developed with NBT/BCIP (Thermo Fisher Scientific) for 2–10 minutes, and reactions were stopped by washing plates with distilled water and allowed to dry overnight before counting. Images were captured with Immunocapture 6.4 software (Cellular Technology Ltd.), and spots were manually counted.
3
Quantifying SARS-CoV-2 Spike-Specific Antibodies
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MBC stimulations were performed on PBMCs collected from subjects in the convalescent cohort. To induce MBC differentiation into antibody secreting cells, 1x106 PBMCs were stimulated with 10 ng/ml Lectin Pokeweed Mitogen (Sigma-Aldrich), 1/100,000 Protein A from Staphylococcus aureus, Cowan Strain (Sigma-Aldrich), and 6 µg/ml CpG (Invitrogen) in complete RPMI in an incubator at 37µC/5% CO2 for 5 days. After stimulation, cells were counted and added to ELISpot white polystyrene plates (Thermo Fisher) coated with 4 µg/ml of SARS-CoV-2 spike that were blocked with 200 µl of complete RPMI. ELISpot plates were incubated with cells for 16 hours overnight in an incubator at 37°C/5% CO2. After the overnight incubation, plates were washed and incubated with anti-IgG-biotin and/or anti-IgA-biotin (Mabtech) for 2 hours at room temperature. After secondary antibody incubation, plates were washed and incubated with streptavidin-alkaline phosphatase (Southern Biotech) for 2 hours at room temperature. Plates were washed and developed with NBT/BCIP (Thermo Fisher Scientific) for 2–10 minutes, and reactions were stopped by washing plates with distilled water and allowed to dry overnight before counting. Images were captured with Immunocapture 6.4 software (Cellular Technology Ltd.), and spots were manually counted.
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