Flow cytometry was performed as previously described [23 (link)–25 (link)]. Briefly, peripheral blood mononuclear cells were collected from immunized volunteers at 0 and 7 days post-vaccination. Day 7 post-vaccination is when the peripheral blood B cell response peaks allowing analysis [23 (link),28 (link)]. Enriched lymphocytes were blocked with PPS22F and CWPS to prevent non-specific labeling with fluorescent PPS. Cells were then labeled with 10 μg/ml of either PPS14-CB or PPS23F-5-DTAF. Fluorochrome-conjugated monoclonal antibodies (BD Bioscience or eBioscience) to the following anti-human Ags were used: CD19 (APC- Cy7), CD27 (PerCP- Cy5.5), IgM (APC). Cells were analyzed as previously described with FACSAria using FACSDiva software (BD Biosciences) [23 (link),24 (link)]. FCS files were analyzed using FlowJo software (Tree Star, Ashland, OR). Populations were divided into three sub- populations: naïve (CD27−), IgM+ memory (CD27+IgM+), and switched memory (CD27+IgM−) B cells. Absolute numbers of B cells were calculated for each donor individually by multiplying the lymphocyte counts by the percentage of B cells determined by flow cytometry.
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Flow Cytometric Analysis of Vaccine-induced B Cell Response
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B cells
Cb 10
Cells
Cy5 5
Donor
Dtaf
Flow cytometry
Fluorochrome
Human
Lymphocyte
Memory
Monoclonal antibodies
Peripheral blood cell
Peripheral blood mononuclear cells
Populations
Tree
Vaccination
Volunteers
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Other organizations : University of Toledo
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Variable analysis
independent variables
- Time post-vaccination (0 days and 7 days)
- Peripheral blood B cell response
- Absolute numbers of B cells
- Percentages of naïve (CD27-, IgM+), IgM+ memory (CD27+IgM+), and switched memory (CD27+IgM-) B cells
- Peripheral blood mononuclear cells collected from immunized volunteers
- Enriched lymphocytes blocked with PPS22F and CWPS to prevent non-specific labeling
- Fluorochrome-conjugated monoclonal antibodies (CD19, CD27, IgM) used for labeling
- No positive or negative controls were explicitly mentioned in the provided information.
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