A comparison of hydraulic and laser capture microdissection methods for collection of single B cells, PCR, and sequencing of antibody VDJ.
During the development of B lymphocytes, a series of gene rearrangements assemble the sequences that encode immunoglobulin heavy and light chains (VDJ). Earlier studies of VDJ sequence diversification during expansion of cells in splenic or appendix germinal centers used hydraulic micromanipulation (HM) to collect single B cells for PCR amplification of rearranged antibody heavy and light chain genes. PCR products were directly sequenced without a cloning step. Hydraulic micromanipulation is a very tedious method. Once capability to collect single cells by laser capture microdissection (LCM) was developed, we modified previous tissue staining and fixation methods so that we could collect cells from a given stained tissue section by HM and LCM and directly compare our success rates using these two methods. Cells were alkaline lysed and after two rounds of nested PCR products were recovered and directly sequenced. Because each rearrangement of genomic DNA that occurs to form the immunoglobulin heavy-chain-encoding sequence in developing B cells is unique, this system allowed us to verify our success rate in recovering single lymphocytes from tissue sections and amplifying a single allele. The methods developed have now made LCM an efficient alternative to HM for the collection of single B cells.
Obiakor
Harold
H
Laboratory of Immunology, National Institutes of Health, Bethesda, Maryland 20892, USA.
Sehgal
Devinder
D
Dasso
Joseph F
JF
Bonner
Robert F
RF
Malekafzali
Arash
A
Mage
Rose G
RG
D000818:Animals; D000916:Antibody Diversity / Q000235:genetics; D001065:Appendix / Q000378:metabolism; D001402:B-Lymphocytes / Q000166:cytology* / Q000378:metabolism*; D001483:Base Sequence; D002469:Cell Separation / Q000295:instrumentation* / Q000379:methods; D017931:DNA Primers; D004210:Dissection / Q000295:instrumentation* / Q000379:methods; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics*; D007147:Immunoglobulin Light Chains / Q000235:genetics*; D007834:Lasers; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011817:Rabbits; D017422:Sequence Analysis, DNA / Q000379:methods*; D013154:Spleen / Q000378:metabolism; D014867:Water
D017931:DNA Primers; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D014867:Water