细胞冻存对抗原表达的影响

niwanmao

相信很多朋友在遇到珍贵标本时，而手头的抗体检测不全时，首先想到的都是把它冻存起来，以后做进一步的检测。这方法可能对分子生物学可能影响不大，但对于流式，可能就影响很大。各位站友可以在此一起讨论一下，在标本保存方面有什么经验？

Cytometry B Clin Cytom. 2012 Jan;82(1):54-8. doi: 10.1002/cyto.b.20621. Epub 2011 Sep 20.
Cryopreservation modulates the detection of regulatory T cell markers.
Sattui S, de la Flor C, Sanchez C, Lewis D, Lopez G, Rizo-Patrón E, White AC Jr, Montes M.
Universidad Peruana Cayetano Heredia, Lima, Peru.

BACKGROUND: Regulatory T cells (Tregs) modulate the host response in infectious diseases and are key mediators of peripheral tolerance. Cryopreservation of peripheral blood mononuclear cells (PBMCs) is commonly used in immunological field studies where access to complex laboratory tests is not feasible. Our objective is to assess the effects of cryopreservation on the flow cytometric detection of surface and intracellular markers of Tregs.
METHODS: Heparinized venous blood was obtained from 36 healthy individuals and 15 HIV-1 infected subjects. PBMCs were isolated and stained for surface and intracellular markers of Tregs. PBMCs from each subject were cryopreserved in liquid nitrogen with DMSO; these cells were thawed and stained at a later date. All samples were analyzed by flow cytometry. The proportion of Tregs was compared using Wilcoxon signed-rank test.
RESULTS: Cryopreservation decreased the proportion of Tregs identified by surface and intracellular markers in healthy individuals and in HIV-1 patients. The proportion of CD4+CD25+FoxP3+ was decreased from 3.13 to 2.16% (P < 0.001) for non-HIV subjects and from 2.68 to 0.94% (P < 0.001) for HIV subjects, compared to fresh samples.（与新鲜标本相比，冻存后复苏的细胞其Treg比例明显降低） Significant reduction was also observed for CD4+CD25+CD127lo-neg.  However, the effect varied considerably between samples. The effect was similar
among HIV and non-HIV patients (P = 0.38).
CONCLUSIONS: Cryopreservation modulates the detection of surface and intracellular markers of Tregs. These results confirm that research on Tregs, including studies of HIV-1 infected patients, should be carried out prospectively on fresh samples in order to obtain unbiased conclusions. Results using cryopreserved cells should be regarded as only preliminary.

PMID: 21936048  [PubMed - indexed for MEDLINE]

－－－－－－－－－－－－其它参考文献－－－－－－－－－－－－－－
Costantini,A., Mancini,S., Giuliodoro,S., Butini,L., Regnery,C.M., Silvestri,G., and Montroni,M. (2003). Effects of cryopreservation on lymphocyte immunophenotype and function. J. Immunol. Methods *278*, 145-155.

Deneys,V., Thiry,V., Hougardy,N., Mazzon,A.M., Leveugle,P., and De,B.M. (1999). Impact of cryopreservation on B cell chronic lymphocytic leukaemia phenotype. J. Immunol. Methods *228*, 13-21.

Pinto,L.A., Trivett,M.T., Wallace,D., Higgins,J., Baseler,M., Terabe,M., Belyakov,I.M., Berzofsky,J.A., and Hildesheim,A. (2005). Fixation and cryopreservation of whole blood and isolated mononuclear cells: Influence of different procedures on lymphocyte subset analysis by flow cytometry. Cytometry B Clin. Cytom. *63*, 47-55.

Rosillo,M.C., Ortuno,F., Rivera,J., Moraleda,J.M., and Vicente,V. (1995). Cryopreservation modifies flow-cytometric analysis of hemopoietic cells. Vox Sang. *68*, 210-214.

Vondracek,J., Soucek,K., Sheard,M.A., Chramostova,K., Andrysik,Z., Hofmanova,J., and Kozubik,A. (2006). Dimethyl sulfoxide potentiates death receptor-mediated apoptosis in the human myeloid leukemia U937 cell line through enhancement of mitochondrial membrane depolarization. Leuk. Res. *30 *, 81-89.

ambassador

一般放4°放半个小时，会有影响吗？有时候约了流式，可是前面拖了时间，只能先放4°。。

niwanmao

ambassador 发表于 2012-12-11 00:30:36

                               
登录/注册后可看大图

一般放4°放半个小时，会有影响吗？有时候约了流式，可是前面拖了时间，只能先放4°。。

一般来说，一个小时内影响不大，当然，越早越好。

刘星宇

血液标本室温存放24小时应该影响不大吧？

niwanmao

刘星宇 发表于 2013-01-07 17:29:22

                               
登录/注册后可看大图

血液标本室温存放24小时应该影响不大吧？

没有染色之前问题不大。