通过抗人 CD14 mAb 和自动化磁激活细胞分选增强马外周血 CD14+细胞的富集方案。

Enhanced protocol for CD14+ cell enrichment from equine peripheral blood via anti-human CD14 mAb and automated magnetic activated cell sorting.

机构信息

Equine Clinic, University of Veterinary Medicine Hannover, Hannover, Germany.

出版信息

Equine Vet J. 2013 Mar;45(2):249-53. doi: 10.1111/j.2042-3306.2012.00616.x. Epub 2012 Sep 19.

DOI:10.1111/j.2042-3306.2012.00616.x

PMID:22994596

Abstract

REASONS FOR PERFORMING STUDY

CD14 positive (CD14+) cells are the precursor cells of monocyte-derived dendritic cells (DCs). In horses their potent antigen-presenting capacity and ability to induce an effective immune response classify these cells suitable for several therapeutic approaches such as for equine sarcoid. However, in horses, the generation efficiency of DCs from adherent peripheral blood mononuclear cells (PBMCs) is currently still poor.

OBJECTIVES

Establishment of a simple short protocol to enhance DC generation in horses by using a human CD14 monoclonal antibody (mAb) and an automated magnetic activated cell sorting (MACS) system.

METHODS

Peripheral blood mononuclear cells were isolated from fresh heparinised blood samples of 3 horses and primarily stained for flow cytometric analysis (FACS) with a mAb against human CD14 as well as a secondary phycoerythrin (PE) conjugated antibody to determine the initial percentage of CD14 cells in the sample. Peripheral blood mononuclear cells were used for automated MACS using the same primary and secondary antibodies and analysed by FACS. CD14+ selected cells were cultured for 4 days adding granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) to the culture media. Dendritic cell generation was assessed analysing cell morphology and surface marker expression (hCD83, hCD86, eqMHCII).

RESULTS

Prior to selection, the mean percentage of CD14+ cells in the total cell population was 5.5%, further gaiting of this cell population resulted in 78.46% CD14+ monocytes. After our positive selection the mean percentage of CD14+ cells in the population was 98% without affecting viability. After culture, DC yield was 2-fold higher than in previous published outcomes.

CONCLUSIONS

The additional CD14 cell separation step after PBMC isolation significantly amplified the number of CD14+ cells, increasing the number of generated DCs.

POTENTIAL RELEVANCE

The number of DCs available is critical for further use of these cells and the herein described protocol will therefore help to improved DC generation for therapeutic approaches in horses.

摘要

研究目的

CD14 阳性（CD14+）细胞是单核细胞来源树突状细胞（DC）的前体细胞。在马中，这些细胞具有强大的抗原呈递能力和诱导有效免疫反应的能力，使它们适合于几种治疗方法，如马的肉瘤。然而，目前马从贴壁外周血单核细胞（PBMC）中生成 DC 的效率仍然很低。

研究目的

建立一种使用人 CD14 单克隆抗体（mAb）和自动化磁性激活细胞分选（MACS）系统增强马 DC 生成的简单短方案。

方法

从 3 匹马的新鲜肝素化血液样本中分离外周血单核细胞，并通过流式细胞术分析（FACS）用针对人 CD14 的 mAb 以及与二级藻红蛋白（PE）偶联的抗体对其进行初步染色，以确定样本中初始 CD14 细胞的百分比。使用相同的初级和二级抗体对外周血单核细胞进行自动化 MACS，并通过 FACS 进行分析。将 CD14+选择的细胞培养 4 天，在培养基中添加粒细胞-巨噬细胞集落刺激因子（GM-CSF）和白细胞介素-4（IL-4）。通过分析细胞形态和表面标记物表达（hCD83、hCD86、eqMHCII）来评估树突状细胞的生成。