A simplified method for Ca2+ flux measurement on isolated human B cells that uses flow cytometry

L. Gergely, Linda Cook, Vincent Agnello

Research output: Contribution to journalArticle

23 Citations (Scopus)

Abstract

A method for Ca2+ flux measurement on isolated human peripheral B cells that uses flow cytometry is described. B cells were isolated by anti-CD19 magnetic bead sorting, and Ca2+ flux was measured with the fluo-3 reagent on a standard single-laser flow cytometer. The response of B-cell stimulation by anti-immunoglobulin B (anti-IgM), anti-IgD, protein A, concanavalin A, and ionomycin was determined. Percentage of responder B cells, the level of Ca2+, and the time of peak stimulation were measured. Bound anti-CD19 monoclonal antibody coupled with small paramagnetic particles did not affect Ca2+ flux. All the isolated B cells responded maximally at 10 s with stimulation by 8 μg of ionomycin. The average isolated preparation contains 70% IgM+ and 85% IgD+ cells, all of which showed peak stimulation with 10 μg of anti-IgM and anti-IgD per ml, respectively, at 30 s. Only at high concentrations of 80 μg/ml, concanavalin A produced a slower response, peaking at 90 s after stimulation. Stimulation with 20 μg of protein A per ml resulted in Ca2+ flux in only 40 to 60% of cells that had a rapid response and maximal stimulation resembling the pattern of activation of ionomycin. B cells from three patients with mixed cryoglobulinemia with high concentrations of monoclonal rheumatoid factors showed stimulation with aggregated IgG, whereas those from healthy control subjects did not, demonstrating the applicability of the methodology to detection of specific antigen stimulation of B cells. This methodology may be useful in testing the functional capacity of B cells in a variety of diseases. The methodology may also prove useful in studying antigen-specific B-cell responses when they involve a significant percentage of B cells.

Original languageEnglish
Pages (from-to)70-74
Number of pages5
JournalClinical and Diagnostic Laboratory Immunology
Volume4
Issue number1
Publication statusPublished - 1997

Fingerprint

Flow cytometry
Flow Cytometry
B-Lymphocytes
Cells
Fluxes
Ionomycin
Staphylococcal Protein A
Concanavalin A
Antigens
Cryoglobulinemia
Immunoglobulin D
Rheumatoid Factor
Sorting
Immunoglobulin M
Healthy Volunteers
Lasers
Immunoglobulin G
Chemical activation
Monoclonal Antibodies

ASJC Scopus subject areas

  • Clinical Biochemistry
  • Immunology
  • Immunology and Allergy
  • Microbiology (medical)

Cite this

A simplified method for Ca2+ flux measurement on isolated human B cells that uses flow cytometry. / Gergely, L.; Cook, Linda; Agnello, Vincent.

In: Clinical and Diagnostic Laboratory Immunology, Vol. 4, No. 1, 1997, p. 70-74.

Research output: Contribution to journalArticle

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