Cryopreservation of peripheral blood mononuclear cells does not significantly affect the levels of spontaneous apoptosis after 24-h culture.
Studies performed with malaria patients living in endemic areas are frequently conducted in laboratories located hundreds of kilometer away from research centers, due to the difficulties in performing the assays in field conditions. Thus, we considered the potential indication of cryopreservation of peripheral blood mononuclear cells (PBMC), in most fieldwork, and decided to evaluate the effect of cryopreservation of PBMC on spontaneous apoptosis. The membrane integrity of PBMC was tested using three previously described protocols of cryopreservation. Cell samples were obtained from 19 healthy volunteers. Percentage of apoptotic nuclei in short-term PBMC cultures was determined by a sensitive method using 7-aminoactinomycin D followed by flow cytometry. Our results indicate that although cryopreservation can to some extent affect lymphocyte membrane integrity rates, flow cytometry analysis showed that frequencies of spontaneous apoptosis in cryopreserved cells were not significantly modified after 24-h culture. It is concluded that cryopreserved PBMC could be used for measuring spontaneous apoptosis and therefore, could be employed for the study of populations living in areas distant from research centers, allowing the comparative evaluation of samples obtained at different time.
Riccio
Evelyn Kety Pratt
EK
Department of Immunology, WHO Collaborating Center for Research and Training in Immunology of Parasitic Diseases, Instituto Oswaldo Cruz Fiocruz, RJ, Brazil.
Neves
Ivan
I
Banic
Dalma Maria
DM
Corte-Real
Suzana
S
Alecrim
Maria das Graças
Md
Morgado
Mariza
M
Daniel-Ribeiro
Cláudio Tadeu
CT
Ferreira-da-Cruz
Maria de Fátima
Mde F
D017209:Apoptosis; D002462:Cell Membrane / Q000502:physiology; D015925:Cryopreservation / Q000379:methods*; D053938:DNA Fragmentation; D006801:Humans; D066298:In Vitro Techniques; D007963:Leukocytes, Mononuclear / Q000166:cytology* / Q000502:physiology; D008854:Microscopy, Electron; D013997:Time Factors