运行流式细胞术(FC)，尤其是流式荧光激活细胞分选 (FACS)，对于新的研究人员来说可能比较棘手。以下提示和技巧将可以帮助您的团队在进行 FC 和 FACS 时节省时间和精力，并提高整个实验和分析过程的信心。

        Running flow cytometry, especially FACS, or fluorescence-activated cell sorting, can be tricky for new scientists. The following tips and tricks will help save your team time and effort when performing flow cytometry and FACS, while also improving confidence throughout the experiment and analysis processes.

 

熟悉您的细胞仪/ Familiarize Yourself with Your Cytometer(s)

准备您的流式细胞术实验的第一步是确认

（1）您是否可以使用传统流式细胞仪或光谱流式细胞仪。

（2）哪种仪器最适合您的项目。光谱流式细胞仪非常适合对大型多色面板进行高通量表征，但成本可能更高。

（3）了解您选择的仪器上可用的激光器。更大的面板将需要更多的激光器，或面板需要的最少荧光团溢出量。

（4）考虑您选择的仪器有哪些生物安全要求。如果您的样本在分析前无法固定，则抽样过程中的雾化和扩散风险将决定需要采取什么样的安全措施。

The first step in preparing for your cytometry experiment is determining (1) whether you have access to a conventional flow cytometer or spectral flow cytometer. (2) which instrument is most appropriate for your project. Spectral flow cytometers are ideal for high-throughput characterizations of large, multicolor panels but can be more costly. (3) what lasers are available on your instrument of choice. More lasers will be required for larger panels, or panels requiring minimal fluorophore spillover. (4) consider what biosafety requirements are in place for your cytometer of interest. If your samples cannot be fixed prior to analysis, the risk of aerosolization during sample aspiration will determine what safety measures will be necessary.

 

有效地设计您的面板/ Designing Your Panel Efficiently

        如果您正在为流式细胞术或细胞分选设计多色面板，单色控件也是必须的。在这些情况下，可以使用抗体结合珠或单色染色对照来完成补偿。如有必要，也可以使用荧光减一 (FMO) 对照，尤其是在您第一次测试新标记时。最佳实践包括为低表达细胞表面标记选择更亮的荧光团（即 PE），反之亦然。然而，串联染料会导致供体荧光团的信号溢出到其他通道中，因此请确保在开始之前绘制整个面板的荧光图。

If you are designing a multicolor panel for your flow cytometry or cell sort, single-color controls are also a must. In these cases, compensation can be completed using either antibody binding beads or single-color stained controls. If necessary, fluorescence minus one (FMO) controls can also be used, particularly for your first time testing a new marker. Best practice involves choosing brighter fluorophores (i.e. PE) for lower expression cell surface markers and vice versa. Tandem dyes, however, can cause signal spillover of the donor fluorophore into other channels, so make sure to map the fluorescence of your entire panel before beginning.

 

完成试点研究/ Complete a Pilot Study

       使用正确滴定的染色抗体浓度对于成功的多色流式面板至关重要。对于细胞分选，考虑到更严格的门控实践，试点研究将确保可以从您的大型实验中获得足够的细胞数量。

Using properly titrated staining antibody concentrations is essential for successful multi-color flow panels. For cell sorting, given stricter gating practice, a pilot study will ensure sufficient cell numbers can be obtained from your larger experiment.

 

考虑信号退化/ Consider Signal Degradation

一些细胞分选项目需要在分选后培养细胞。然而，根据目标细胞类型，荧光团可能会在培养物中保持结合。

这些荧光团会导致：

（1）在后续的实验中影响这些标记的检测；

（2） 由于抗体结合而干扰细胞信号传导；

（3）阻止细胞标记或其他细胞受体与配体结合；

（4）荧光团可以降解或解耦和干扰与其他荧光团。

出于这些原因，细胞分选面板应尽量减少荧光团以达到仅包含必要的标记。

Some cell sorting projects require culturing of cells following sorting. However, depending on the target cell type(s), fluorophores may remain bound in culture. This can 1) effect detection of those markers in future experiments； 2) interfere with cell signaling because of antibody binding；3) block cell markers or other cell receptors from ligand binding； 4) the fluorophores can degrade or decouple and interfere with other fluorophores. For these reasons, cell sorting panels should be minimized to include only necessary markers.

 

在分析之前富集稀有细胞类型/ Enrich Rare Cell Types Prior to Analyzing

        无论您是运行标准流式细胞术还是通过 FACS 进行细胞分选，稀有细胞类型都会导致额外的试剂浪费和仪器上不必要的时间。获得更高频率的目标群体的一种方法是使用磁珠细胞分离，以便为您感兴趣的子集进行消极选择。通过在分析之前富集您的样本，将提高为您的项目捕获可靠数量的稀有细胞的可能性。

Whether you are running standard flow cytometry or cell sorting by FACS, rare cell types can lead to additional reagent waste and unnecessary time at the instrument. One way to achieve a higher frequency of your target population is to use magnetic bead cell separation in order to negatively select for your subset of interest. By enriching your samples prior to analyzing, you will improve the likelihood of catching rare cells in reliable numbers for your project(s).

 

        如果您在设计流式细胞仪或 FACS 实验时有任何问题，请随时联系 Biorbyt 团队以获取有关实验的建议。同时，Biorbyt 可以提供各种常用靶标的流式抗体，比如CD4，CD3，CD8，CD38，CD19等，欢迎咨询！

If you have any questions designing your flow cytometry or FACS experiments, please feel free to contact the Biorbyt team for advice on your experiments. At the same time, Biorbyt can provide FC& FACS antibodies for various commonly used targets, such as CD4, CD3, CD8, CD38, CD19, etc. Welcome to consult!

 

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