He equation y = 10 (0.1344x+0.5415). (C) The proportion of viable cells during long-term culture was monitored on a regular basis for each culture. (D) The mean value and standard deviation of viability ( ) showed a similar evolution for each samples. doi:10.1371/journal.pone.0051946.gthe end of the culture period by RT-PCR amplification of EBNA1, as previously published [16].Mini-scale CulturesWhen indicated, switched-memory B cell cultures were initiated as described above in 24- or 6-well plates in the presence of cirradiated L4.5 cells up to a total number of 4 to 56106 cells for their subsequent transfer into a Petri dish (100620 mm, BD Biosciences). Cells were seeded at a final concentration of 36105 cells/mL, in a final volume of 15 mL per Petri dish, and expanded to obtain a final volume of approximately 0.5 L.monoclonal antibodies obtained from BD Biosciences. FITCconjugated anti-IgM (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) and anti-IgA (AbD Serotec, Raleigh, NC, USA) were polyclonal goat antibodies. Cells were stained and fixed with 2 paraformaldehyde (Sigma-Aldrich, Oakville, ON, Canada). All analyses were done on 5000 to 10000 viable cells gated in a region determined by 7-amino-actinomycin-D (7AAD, BD Biosciences). Analyses were performed using a FACSCalibur flow Title Loaded From File cytometer and the CellQuest software (BD Biosciences). Data were subsequently analyzed using FCS Express II (De Novo Software, Los Angeles, CA, USA).Flow Cytometry AnalysesAPC-conjugated anti-CD14, anti-CD19, anti-CD38 and antiIgG, FITC-conjugated anti-CD3, anti-IgM and anti-IgA, PEconjugated anti-CD45, anti-CD138 and anti-IgD, and PerCPCy5.5-conjugated anti-CD3 and anti-CD19, were IgG1 mouseDetermination of Immunoglobulin ConcentrationsIgA, IgG, IgG1, IgG2, IgG3 IgG4 and IgM concentrations in culture supernatants were determined by ELISA. Goat affinitypurified antibodies specific to human Fc fragment of IgA, IgG, IgM (Jackson ImmunoResearch Laboratories), IgG1 and IgG3 (Invitrogen), IgG2 (BD Biosciences) and IgG4 (Southern Biotech,Large-Scale Expansion of Human B LymphocytesBirmingham, USA) were used to capture the secreted immunoglobulins. IgG and IgM were revealed using peroxidase-conjugated goat antibodies against human Ig (Jackson ImmunoResearch Laboratories). IgA were revealed with peroxidase-conjugated goat anti-human antibodies specific to the alpha chain while IgG subclasses were revealed using goat anti-human antibodies specific for the gamma chain of the Fc fragment. IgE concentrations were determined using Human IgE Ready-SET-Go following manufacturer’s instructions (eBioscience Inc., San Diego, USA), Secretion rates were determined using washed cells seeded at 26106cells/ml for 20 to 22 hours without cytokines or L4.5 cells. This culture’s supernatant was then used in an ELISA assay as described above.focusing process (Bio-Rad Laboratories, Mississauga, Canada). According to a standard western blot assay, proteins were transferred from gels to Amersham Hybond-ECL nitrocellulose (GE Healthcare, Piscataway, USA) and membranes were revealed using peroxidaseconjugated goat antibodies specific to human c Title Loaded From File chains (Jackson ImmunoResearch Laboratories). Detection was done with the Amersham ECLTM Western blotting detection reagents chemiluminescence kit (GE Healthcare), following 18325633 the manufacturer’s instructions.Human Protein MicroarrayThe reactivity of the in vitro generated human IgG was determined by antibody specificity profiling service of.He equation y = 10 (0.1344x+0.5415). (C) The proportion of viable cells during long-term culture was monitored on a regular basis for each culture. (D) The mean value and standard deviation of viability ( ) showed a similar evolution for each samples. doi:10.1371/journal.pone.0051946.gthe end of the culture period by RT-PCR amplification of EBNA1, as previously published [16].Mini-scale CulturesWhen indicated, switched-memory B cell cultures were initiated as described above in 24- or 6-well plates in the presence of cirradiated L4.5 cells up to a total number of 4 to 56106 cells for their subsequent transfer into a Petri dish (100620 mm, BD Biosciences). Cells were seeded at a final concentration of 36105 cells/mL, in a final volume of 15 mL per Petri dish, and expanded to obtain a final volume of approximately 0.5 L.monoclonal antibodies obtained from BD Biosciences. FITCconjugated anti-IgM (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) and anti-IgA (AbD Serotec, Raleigh, NC, USA) were polyclonal goat antibodies. Cells were stained and fixed with 2 paraformaldehyde (Sigma-Aldrich, Oakville, ON, Canada). All analyses were done on 5000 to 10000 viable cells gated in a region determined by 7-amino-actinomycin-D (7AAD, BD Biosciences). Analyses were performed using a FACSCalibur flow cytometer and the CellQuest software (BD Biosciences). Data were subsequently analyzed using FCS Express II (De Novo Software, Los Angeles, CA, USA).Flow Cytometry AnalysesAPC-conjugated anti-CD14, anti-CD19, anti-CD38 and antiIgG, FITC-conjugated anti-CD3, anti-IgM and anti-IgA, PEconjugated anti-CD45, anti-CD138 and anti-IgD, and PerCPCy5.5-conjugated anti-CD3 and anti-CD19, were IgG1 mouseDetermination of Immunoglobulin ConcentrationsIgA, IgG, IgG1, IgG2, IgG3 IgG4 and IgM concentrations in culture supernatants were determined by ELISA. Goat affinitypurified antibodies specific to human Fc fragment of IgA, IgG, IgM (Jackson ImmunoResearch Laboratories), IgG1 and IgG3 (Invitrogen), IgG2 (BD Biosciences) and IgG4 (Southern Biotech,Large-Scale Expansion of Human B LymphocytesBirmingham, USA) were used to capture the secreted immunoglobulins. IgG and IgM were revealed using peroxidase-conjugated goat antibodies against human Ig (Jackson ImmunoResearch Laboratories). IgA were revealed with peroxidase-conjugated goat anti-human antibodies specific to the alpha chain while IgG subclasses were revealed using goat anti-human antibodies specific for the gamma chain of the Fc fragment. IgE concentrations were determined using Human IgE Ready-SET-Go following manufacturer’s instructions (eBioscience Inc., San Diego, USA), Secretion rates were determined using washed cells seeded at 26106cells/ml for 20 to 22 hours without cytokines or L4.5 cells. This culture’s supernatant was then used in an ELISA assay as described above.focusing process (Bio-Rad Laboratories, Mississauga, Canada). According to a standard western blot assay, proteins were transferred from gels to Amersham Hybond-ECL nitrocellulose (GE Healthcare, Piscataway, USA) and membranes were revealed using peroxidaseconjugated goat antibodies specific to human c chains (Jackson ImmunoResearch Laboratories). Detection was done with the Amersham ECLTM Western blotting detection reagents chemiluminescence kit (GE Healthcare), following 18325633 the manufacturer’s instructions.Human Protein MicroarrayThe reactivity of the in vitro generated human IgG was determined by antibody specificity profiling service of.
