血液和脊髓免疫细胞流式圈门分析解决方案

流式细胞术分析的核心在于“圈门"，所谓“门"和“区域"，是指基于特定特征对细胞群体进行划分，常通过FSC（前向散射）、SSC（侧向散射）及表面标记物（marker）来进行“圈门"，以分辨和量化不同的细胞群。圈门的第一步通常是根据细胞的FSC和SSC特性来区分不同的细胞群。FSC反映了细胞的大小，SSC则与细胞的粒度相关。同时这些信号也会受到样本预处理、激光波长、采集角度、折射率以及鞘液的流速等因素的影响。在分析复杂样本（如PBMC外周血单核细胞）时，FSC和SSC的组合有助于区分不同类型的细胞，如淋巴细胞与细胞碎片或死细胞。通过调整阈值，便可以有效排除不相关的细胞群体，如死细胞。

PBMC包含多种不同类型的细胞。通过光散射特性，可以将单核细胞和淋巴细胞从细胞碎片和死细胞中区分开细胞碎片和死细胞通常表现出较低的FSC信号，一般位于密度图的左下角区域。为有效排除不相关的细胞，可以通过调整FSC阈值来去除这些细胞，或者通过进一步优化圈门策略，缩小分析范围。这些方法有助于去除由样本中的自身荧光或抗体非特异性结合所引起的背景信号和死细胞。此外，使用死活细胞染料（如DAPI、7-AAD等）也是一种行之有效的手段，可以帮助进一步排除死细胞，确保结果的可靠性。

参考文献的圈门，尤其是血液和脊髓免疫细胞流式圈门分析解决方案，可以参考如下这篇文献。

论文信息：

论文题目：NAAA-regulated lipid signaling in monocytes controls the induction of hyperalgesic priming in mice

期刊名称：Nature Communications

时间期卷：15, Article number: 1705 (2024)

在线时间：2024年2月24日

DOI：doi.org/10.1038/s41467-024-46139-5

产品信息：

货号：CP-005-005

规格：5ml+5ml

品牌：Liposoma

产地：荷兰

名称：Clodronate Liposomes and Control Liposomes

办事处：Target Technology（靶点科技）

血液和脊髓免疫细胞流式圈门分析解决方案：

Fluorescence Activated Cell Sorting (FACS)

Blood was drawn via cardiac puncture and incubated with ammonium-chloride-potassium (ACK) buffer for erythrocyte lysis. Leukocytes were stained with anti-CD11b, Zombie NIR, anti-Ly6C, and anti-Ly6G antibodies (Table S1) in antibody-staining buffer (0.2% BSA and 0.1% sodium azide in PBS) for 30 min at 4 °C. Viable cell populations of interest were collected by FACS and quantified using FCS Express 7 (De Novo Software, Pasadena, CA). Gating strategies are illustrated in Figure S1. First, to separate debris from cells, we gated on the side scatter area (SSC-A) versus the forward scatter area (FSC-A) and selected the cell population with a high FSC-A/SSC-A ratio. This fraction was subjected to forward scatter height (FSC-H) versus FSC-A analysis to isolate single cells that displayed a ratio of ∼1. The single-cell population was further processed to remove dead cells. Zombie NIR signal-negative cells were collected for the next steps. All FACS processes included these initial gating steps to ensure that only viable single cells were analyzed. From this population, we gated cells based on the expression of cellular marker proteins to isolate cell types of interest. To focus on innate immune cells, we selected cells that were positive for CD11b by plotting the viable cell population as CD11b versus FSC-A and selecting the population that was CD11b+. This was plotted as Ly6C versus Ly6G with the Ly6Chigh cells being divided into groups depending on Ly6G expression. Monocytes were categorized as ‘classical’ (CD11b+ Ly6Chigh Ly6Glow) or ‘non-classical’ (CD11b+ Ly6Clow Ly6Glow), while neutrophils were defined as CD11b+ Ly6Chigh Ly6Ghigh . Expression of the Naaa and Ppara genes in monocytes and neutrophils was quantified by reverse transcription-quantitative polymerase chain reaction (RT-qPCR), as described below, to confirm successful recombination.

The spinal cord underwent enzymatic digestion and mechanical dissociation using a brain dissociation kit (Miltenyi Biotec, Bergisch Gladbach, Germany), following the manufacturer’s instructions. Subsequently, the dissociated spinal cord cells were stained with anti-CD11b and anti-CD45 antibodies (Table S1) in antibody-staining buffer (0.2% BSA and 0.1% sodium azide in PBS) for 30 min at 4 °C. Viable cell populations were collected by FACS and quantified using FCS Express version 7.18.0025 (De Novo Software, Pasadena, CA). Gating strategies are described above. Microglia was plotted as CD45 versus CD11b, which are both established markers for this cell lineage.