Cryopreserved human B cells as an alternative source for single cell mRNA analysis

被引：6

作者：

Hansen A. ^{[1
]}

Reiter K. ^{[1
,2
]}

Dörner T. ^{[2
]}

Pruss A. ^{[2
]}

机构：

[1] Outpatients Department of Medicine, Charite University Medicine Berlin, D-10117 Berlin

[2] Institute for Transfusion Medicine (Tissue Bank and Research Unit), Charite University Medicine Berlin, D-10117 Berlin

来源：

Cell and Tissue Banking | 2005年 / 6卷 / 4期

关键词：

Cryopreservation; FACS; Human B cells; mRNA; Single cell RT-PCR;

D O I：

10.1007/s10561-005-8348-2

中图分类号：

学科分类号：

摘要：

Reverse transcription-polymerase chain reaction (RT-PCR) of individual B-lymphocytes has been shown to be a powerful tool for the simultaneous analysis of different mRNA specificities in both malignant and non-malignant B cell subpopulations. However, especially for longitudinal studies, this may also require analyses of cryopreserved cells. Therefore, the current study assessed whether cryopreserved (liquid nitrogen, dimethyl sulfoxide [DMSO] - stored) viable B cells are an alternative source for single cell RT-PCR analysis. Fresh (non-frozen) and post-thawed human peripheral blood B cells were analyzed by fluorescence-activated cell sorting (FACS). As a result, different B cell subpopulations could be reliably stained and separated from both fresh and post-thawed cells by four-color flow cytometry, although slightly diminished fluorescence intensities of some subpopulation markers were observed when analyzing cryopreserved cells. Subsequently, viable individual CD19+CD27+ memory B cells were sorted into single wells and analyzed for the expression of mRNA transcripts of the -house-keeping gene' glyceraldehyde phosphate dehydrogenase (GAPD), the constitutive B cell homing receptor CXCR4, and immunoglobulin heavy chain variable region (IgVH) genes by nested RT-PCR protocols. Comparing both B cell sources, RT-PCR analysis revealed comparable yields of cells expressing transcripts for the three mRNA specificities tested (GAPD, CXCR4, IgVH) indicating the integrity of the respective mRNAs in cryopreserved B cells. In conclusion, these data indicate that optimally cryopreserved B cells may be an alternative source for single-cell RT-PCR analysis, especially in longitudinal B cell studies. However, the settings for both FACS analysis and RT-PCR should be re-evaluated for each distinct subpopulation and target mRNA of interest when analyzing post-thawed cells. © Springer 2005.

引用

页码：299 / 308

页数：9

共 28 条

[1]

Agematsu K., Nagumo H., Yang F.C., Nakazawa T., Fukushima K., Ito S., Sugita K., Mori T., Kobata T., Morimoto T., Komiyama A., B cell subpopulations separated by CD27 and crucial collaboration of CD27+ B cells and helper T cells in immunoglobulin production, Eur. J. Immunol., 27, pp. 2073-2079, (1997)

[2]

Agematsu K., Nagumo H., Oguchi Y., Nakazawa T., Fukushima K., Yasui K., Ito S., Kobata T., Morimoto T., Komiyama A., Generation of plasma cells from peripheral blood memory B cells: Synergistic effect of interleukin-10 and CD27/CD70 interaction, Blood, 91, pp. 173-180, (1998)

[3]

Bohme H., Seifert M., Roggenbuck D., Docke W., von Baehr R., Hansen A., Characterization of a B-CLL derived IgM-λ antibody expressing typical features of a NPAB, Immunol. Lett., 41, pp. 261-266, (1994)

[4]

Celluzzi C.M., Welbon C., A simple cryopreservation method for dendritic cells and cells used in their derivation and functional assessment, Transfusion, 43, pp. 488-494, (2003)

[5]

Dong H.Y., Shahsafaei A., Dorfman D.M., CD148 and CD27 are expressed in B cell lymphomas derived from both memory and naive B cells, Leuk. Lymphoma, 43, pp. 1855-1858, (2002)

[6]

Girschick H.J., Grammer A.C., Nanki T., Vazquez E., Lipsky P.E., Expression of recombination activating genes 1 and 2 in peripheral B cells of patients with systemic lupus erythematosus, Arthritis Rheum., 46, pp. 1255-1263, (2002)

[7]

Hansen A., Jahn S., Lukowsky A., Grutz G., Bohn J., von Baehr R., Settmacher U., VH/VL gene expression in polyreactive-antibody-producing human hybridomas from the fetal B cell repertoire, Exp. Clin. Immunogenet., 11, pp. 1-16, (1994)

[8]

Hansen A., Odendahl M., Reiter K., Jacobi A.M., Feist E., Scholze J., Burmester G.R., Lipsky P.E., Dorner T., Diminished peripheral blood memory B cells and accumulation of memory B cells in the salivary glands of patients with Sjögren's syndrome, Arthritis Rheum., 46, pp. 2160-2171, (2002)

[9]

Hansen A., Gosemann M., Pruss A., Reiter K., Ruzickova S., Lipsky P.E., Dorner T., Abnormalities in peripheral B cell memory of patients with primary Sjögren's syndrome, Arthritis Rheum., 50, pp. 1897-1908, (2004)

[10]

Hansen A., Reiter K., Ziprian T., Jacobi A.M., Gosemann M., Hoffmann M., Scholze J., Lipsky P.E., Dorner T., Dysregulation of chemokine receptor expression and function by B cells of patients with primary Sjögren's syndrome, Arthritis Rheum., 52, pp. 2109-2119, (2005)

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