DNAse treatment following thawing of cryopreserved PBMC is a procedure suitable for lymphocyte functional studies.
Testing freshly isolated PBMC is not practical for immune monitoring analysis in large clinical trials or natural history studies. Thus, cryopreserved PBMC represent a more practical alternative. However, cell clumping is a common problem following thawing of PBMC isolated from blood that was previously transported and stored. Cell clumping leads to loss of cells, and could affect cell function and/or phenotype. The development and validation of procedures that prevent cell clumping and preserve cell function and surface marker expression levels are necessary to allow evaluation of immune function and phenotype in cryopreserved samples from clinical studies. The incorporation of a DNAse treatment step in the standard thawing procedure efficiently avoided clump formation, and did not result in detectable changes in cell viability, expression of standard leukocyte surface markers or two key parameters of immune function, proliferation and cytokine induction in response to a variety of common stimuli. Therefore, this procedure seems suitable for standard immunologic assays.
García-Piñeres
Alfonso J
AJ
HPV Immunology Laboratory, SAIC-Frederick, Inc., NCI-Frederick, Frederick MD, USA.
Hildesheim
Allan
A
Williams
Marcus
M
Trivett
Matthew
M
Strobl
Susan
S
Pinto
Ligia A
LA
D000941:Antigens / Q000494:pharmacology; D000954:Antigens, Surface / Q000032:analysis; D001402:B-Lymphocytes / Q000737:chemistry / Q000166:cytology / Q000378:metabolism; D015496:CD4-Positive T-Lymphocytes / Q000737:chemistry / Q000166:cytology / Q000378:metabolism; D018414:CD8-Positive T-Lymphocytes / Q000737:chemistry / Q000166:cytology / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D015925:Cryopreservation / Q000379:methods*; D016207:Cytokines / Q000378:metabolism; D003851:Deoxyribonucleases / Q000378:metabolism*; D006801:Humans; D007371:Interferon-gamma / Q000378:metabolism; D007378:Interleukins / Q000378:metabolism; D007694:Killer Cells, Natural / Q000737:chemistry / Q000166:cytology / Q000378:metabolism; D007963:Leukocytes, Mononuclear / Q000737:chemistry / Q000166:cytology* / Q000378:metabolism; D008213:Lymphocyte Activation / Q000276:immunology; D008214:Lymphocytes / Q000737:chemistry / Q000166:cytology* / Q000378:metabolism; D009000:Monocytes / Q000737:chemistry / Q000166:cytology / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D000941:Antigens; D000954:Antigens, Surface; D016207:Cytokines; D007378:Interleukins; D007371:Interferon-gamma; D003851:Deoxyribonucleases; D013755:Tetradecanoylphorbol Acetate