Changes in fluorescence intensity of selected leukocyte surface markers following fixation
Changes in fluorescence intensity of selected leukocyte surface markers following fixation
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DOI：
10.1002/cyto.a.20392
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发表时间：
2007-06-01
期刊：
影响因子：
3.7
通讯作者：
Frampton, Mark W.
中科院分区：
文献类型：
--
作者：
Stewart, Judith C.;Villasmil, Michelle L.;Frampton, Mark W.
背景：血液白细胞的免疫表型分析通常涉及在细胞计数分析之前用多聚甲醛固定。然而，细胞类型和标记特异性对固定后荧光强度稳定性的影响尚未得到充分研究。方法：使用一组异硫氰酸荧光素标记的表面标记抗体对人全血进行染色。染色后 0、2、4、6、24、48 和 96 小时通过流式细胞术分析未固定和固定的样品。将荧光测量结果转换为等效可溶性荧光染料的分子进行比较。结果：固定导致 48 小时时前向和侧向散射显着减少，这需要门控调整以实现细胞群的分辨率。固定样品中的自发荧光逐渐增加（单核细胞在 96 小时时增加九倍）。在校正自发荧光后，标记相关荧光的可变减少变得明显。 96 小时时下降的幅度随细胞类型和标记物的不同而变化，从单核细胞上的 CD32 的 5% 到中性粒细胞上的 CD16 的 39%。结论：白细胞染色和固定后荧光强度的变化随细胞类型和表面标记物的不同而变化。应确定每种细胞类型和所用标记物的荧光稳定性，并应考虑固定对细胞自发荧光的混杂影响。 (c) 2007 年国际分析细胞学学会。
Background: Immunophenotyping of blood leukocytes often involves fixation with paraformaldehyde prior to cytometry analysis. However, the influence of cell type and marker specificity on the stability of fluorescence intensity after fixation has not been well studied.Methods: Human whole blood was stained using a panel of fluorescein isothiocyanate-labeled antibodies to surface markers. Unfixed and fixed samples were analyzed by flow cytometry at 0, 2, 4, 6, 24, 48, and 96 h after staining. Fluorescence measurements were converted to molecules of equivalent soluble fluorochrome for comparison.Results: Fixation caused a significant decrease in both forward and side scatter at 48 h which required gating adjustments to achieve resolution of cell populations. The autofluorescence increased progressively in fixed samples (ninefold at 96 h for monocytes). Variable decreases in marker-associated fluorescence became apparent after correction for autofluorescence. The magnitude of the decrease at 96 h varied with cell type and marker, from 5% for CD32 on monocytes to 39% for CD16 on neutrophils.Conclusion: The change in fluorescence intensity following staining and fixation of leukocytes varies with cell type and surface marker. Fluorescence stability should be determined for each cell type and marker used, and the confounding effects of fixation on cell autofluorescence should be considered. (c) 2007 International Society for Analytical Cytology.