Analysis of the germinal center reaction and in vivo long-lived plasma cells.
This chapter describes the analysis of long- and short-lived plasma cells on tissue sections. Mice are immunized with 4-hydroxy-3-nitrophenyl acetyl (NP) coupled to a T-cell-dependent carrier. Antigen-specific germinal center cells and extrafollicular plasma cells are identified by immunohistology on tissue sections. Plasma cells labeled with 5-bromo-2'-deoxyuridine (BrdU) pulses given during different phases of B-cell response are identified on spleen sections. To identify mutated cells originating from cells in germinal centers, antigen-specific cells from spleen sections are isolated by microdissection. From these NP-specific recombined VDJ genes are amplified with family-specific primers by polymerase chain reaction (PCR) for deoxyribonucleic acid (DNA) sequencing. The methods described can be used to characterize origins and life-span of plasma cells in vivo.
Toellner
Kai-Michael
KM
MRC Centre for Immune Regulation, Medical School, University of Birmingham, Birmingham, UK.
Khan
Mahmood
M
Sze
Daniel Man-Yuen
DM
D000818:Animals; D001402:B-Lymphocytes / Q000166:cytology* / Q000276:immunology; D001973:Bromodeoxyuridine / Q000737:chemistry; D002454:Cell Differentiation / Q000276:immunology*; D002645:Chickens / Q000276:immunology; D018858:Germinal Center / Q000166:cytology* / Q000276:immunology; D007114:Immunization; D051379:Mice; D042282:Microdissection; D009154:Mutation; D009596:Nitrophenols / Q000276:immunology; D010648:Phenylacetates; D010950:Plasma Cells / Q000166:cytology* / Q000276:immunology; D016133:Polymerase Chain Reaction; D011947:Receptors, Antigen, B-Cell / Q000276:immunology / Q000378:metabolism; D012756:Sheep / Q000276:immunology; D013154:Spleen / Q000276:immunology*; D006377:T-Lymphocytes, Helper-Inducer / Q000166:cytology / Q000276:immunology
D009596:Nitrophenols; D010648:Phenylacetates; D011947:Receptors, Antigen, B-Cell; C026242:4-hydroxy-5-nitrophenyl acetic acid; D001973:Bromodeoxyuridine