流式細胞術分析的核心在于“圈門”，所謂“門”和“區域”，是指基于特定特征對細胞群體進行劃分，常通過FSC（前向散射）、SSC（側向散射）及表面標記物（marker）來進行“圈門”，以分辨和量化不同的細胞群。圈門的第一步通常是根據細胞的FSC和SSC特性來區分不同的細胞群。FSC反映了細胞的大小，SSC則與細胞的粒度相關。同時這些信號也會受到樣本預處理、激光波長、采集角度、折射率以及鞘液的流速等因素的影響。在分析復雜樣本（如PBMC外周血單核細胞）時，FSC和SSC的組合有助于區分不同類型的細胞，如淋巴細胞與細胞碎片或死細胞。通過調整閾值，便可以有效排除不相關的細胞群體，如死細胞。

PBMC包含多種不同類型的細胞。通過光散射特性，可以將單核細胞和淋巴細胞從細胞碎片和死細胞中區分開細胞碎片和死細胞通常表現出較低的FSC信號，一般位于密度圖的左下角區域。為有效排除不相關的細胞，可以通過調整FSC閾值來去除這些細胞，或者通過進一步優化圈門策略，縮小分析范圍。這些方法有助于去除由樣本中的自身熒光或抗體非特異性結合所引起的背景信號和死細胞。此外，使用死活細胞染料（如DAPI、7-AAD等）也是一種行之有效的手段，可以幫助進一步排除死細胞，確保結果的可靠性。

參考文獻的圈門，尤其是血液和脊髓免疫細胞流式圈門分析解決方案，可以參考如下這篇文獻。

論文信息：

論文題目：NAAA-regulated lipid signaling in monocytes controls the induction of hyperalgesic priming in mice

期刊名稱：Nature Communications

時間期卷：15, Article number: 1705 (2024)

在線時間：2024年2月24日

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

產品信息：

貨號：CP-005-005

規格：5ml+5ml

品牌：Liposoma

產地：荷蘭

名稱：Clodronate Liposomes and Control Liposomes

辦事處：Target Technology（靶點科技）

血液和脊髓免疫細胞流式圈門分析解決方案：

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.