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  • 發布時間:2020-06-01 14:59 原文鏈接: 小鼠骨髓來源樹突狀細胞(BMDC)的培養(七)

    4.  培養方法的選擇

        本文列出迄今為止最常用的三種BMDC培養方法,我們從下表中可以更清楚地看出這三種方法的異同,相信您能根據您的需要作出正確的判斷。

                             

    注:1. 該表是從文獻[8]修改和添加而來;

        2. 這里的Inaba法指的是其原法[2],而不是本文中的改良法。

    【BMDC 培養試劑推薦】
     

    生產商產品名稱產品編號產品規格使用濃度
    PeproTech重組小鼠 GM-CSF315-035μg/20μg/50μg/100μg/250μg/500μg/1mg25-50ng/ml
    PeproTech重組小鼠 IL-4214-145μg/20μg/50μg/100μg/250μg/500μg/1mg10-40ng/ml
    PeproTech重組小鼠 TNF-α315-01A5μg/20μg/50μg/100μg/250μg/500μg/1mg25-50ng/ml
    PeproTech重組小鼠 sCD40L315-155μg/25μg/100μg/250μg/500μg/1mg1μg/ml


    【BMDC 鑒定試劑推薦】
     

    生產商產品名稱產品編號克隆號熒光標記產品規格
    PeproTech(BioGems)抗小鼠 CD11c熒光標記抗體3212N418FITC/PE/APC/PerCP-Cy5.5  /APC-Cy7/PE-Cy725μg/100μg/500μg
    抗小鼠 CD40熒光標記抗體2512HM40-3FITC50μg/100μg/500μg/1000μg
    抗小鼠 CD80熒光標記抗體291216-10A1FITC/PE/APC25μg/100μg/500μg
    抗小鼠 CD86熒光標記抗體8912GL-1FITC/PE/APC/PE-Cy725μg/100μg/500μg
    抗小鼠 MHC II類分子(I-A/I-E)熒光標記抗體86212M5/114.15.2FITC/PE/APC/PE-Cy725μg/100μg/200μg /500μg


        注:用于DC鑒定的表面標志物的表達在流式圖上均呈現單個峰,且多數情況下不能與陰性峰完全區分開來。為避免多色分析時補償調節不好導致結果的不準確性,建議最好用單標,最多用雙標來做DC表面標志的分析,而且必須使用同型對照,而非空白細胞對照來 排除背景染色。

    【參考文獻】

    [1] Steinman RM, Cohn ZA. Identification of a novel cell type in peripheral lymphoid organs of mice. I. Morphology, quantitation, tissue distribution. J. Exp. Med. 1973; 137 (5): 1142–62.

    [2] Inaba K, Steinman RM, et. al. Identification of proliferating dendritic cell precursors in mouse blood. J. Exp. Med. 1992; 175(5):1157-67.

    [3] Inaba K, Inaba M, et. al. Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulating factor. J. Exp. Med. 1992; 176(6):1693-702.

    [4] Romani N, Gruner S, et. al. Proliferating dendritic cell progenitors in human blood. J. Exp.

    Med. 1994; 180(1):83-93.

    [5] Zorina T, Mayordomo JI, et. al. Culture of dendritic cells from murine bone marrow supplemented with GM-CSF and TNF-alpha J. Immunother. 1994; 16(3):247.

    [6] Mayordomo JI, Zorina T, et. al. Bone marrow-derived dendritic cells pulsed with synthetic tumour peptides elicit protective and therapeutic antitumour immunity. Nat Med. 1995;1(12):1297-302.

    [7]  Labeur MS,  Roters B,  et. al. Generation of  tumor immunity by  bone  marrow-derived dendritic  cells  correlates  with  dendritic  cell  maturation  stage.  J   Immunol.  1999;162(1):168-75.

    [8] Lutz MB, Kukutsch N, et. al. An advanced culture method for generating large quantities of highly  pure  dendritic  cells  from  mouse  bone  marrow.  J  Immunol  Methods.  1999;223(1):77-92.

    [9] Son YI, Egawa S, et. al. A novel bulk-culture method for generating mature dendritic cells from mouse bone marrow cells. J Immunol Methods. 2002; 262(1-2):145-57.

    [10] Inaba, K., Romani, N., et al. Generation of dendritic cells from proliferating mouse bone marrow progenitors. In: Coico, R., Ranz, A., Kruisbeek,A.M. (Eds.), Current Protocols in Immunology.Wiley, New York. 1998; Unit 3.7.


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