AutoSpill: a method for calculating spillover coefficients in high-parameter flow cytometry

Biological utility of AutoSpill. Downstream analyses of data compensated by either the traditional compensation algorithm or AutoSpill. (a) Plots were prepared and compensated using FlowJo v.10.6, using either the default traditional algorithm or uploading the spillover matrix generated by AutoSpill. Representative flow cytometry plots illustrating errors corrected by AutoSpill (first and second column, MM3 dataset; third and fourth column, MM2 dataset). (b-e) All plots were prepared from the same FCS files and compensated using FlowJo v.10.7, using either the traditional algorithm or the AutoSpill option. (b) Hierarchical gating for CD4+CD8+CD25+ lymphocytes, using data compensated by the traditional algorithm or AutoSpill (MM3 dataset). (c) The CD4+CD25+ population was backgated to identify the source of population loss in the traditional algorithm (MM3 dataset). (d) MHCII expression on known negative cells (CD4 T cells), known positive cells (CD11b+ splenocytes), and microglia (MM4 dataset). Percent positive was thresholded using CD4 T cells as the negative. MHCII knockout microglia were used as a “true negative" staining control. (e) Foxp3GFP expression on known bimodal cells (CD4+ splenocytes) and CD11b+ macrophages (MM5 dataset). The positive population was thresholded using the negative CD4 T cell peak. Wildtype mice, without the GFP transgene, were used as a “true negative" staining control. This dataset includes the spillover matrices (traditional and AutoSpill) used in this analysis.

AutoSpill的生物学应用效能。分别采用传统补偿算法与AutoSpill对数据进行补偿后的下游分析。
(a) 本部分散点图通过FlowJo v.10.6制作并完成补偿，分别采用默认传统补偿算法，或导入AutoSpill生成的荧光补偿矩阵（spillover matrix）。代表性流式细胞术散点图展示了AutoSpill校正后的实验误差（第一、二列为MM3数据集；第三、四列为MM2数据集）。
(b-e) 本部分所有散点图均来自同一批FCS格式文件（FCS file），并通过FlowJo v.10.7完成补偿，分别采用传统补偿算法或AutoSpill补偿模式。
(b) 针对CD4+CD8+CD25+淋巴细胞的分层设门分析，分别使用传统补偿算法与AutoSpill补偿后的数据集（MM3数据集）。
(c) 对CD4+CD25+细胞群进行反向设门，以定位传统补偿算法下细胞群丢失的来源（MM3数据集）。
(d) 检测已知阴性细胞（CD4 T细胞）、已知阳性细胞（CD11b+脾细胞）以及小胶质细胞中主要组织相容性复合体II类分子（MHCII）的表达水平（MM4数据集）。以CD4 T细胞作为阴性对照设定阳性表达阈值，采用MHCII基因敲除的小胶质细胞作为染色实验的“真阴性”对照。
(e) 检测已知双峰细胞群（CD4+脾细胞）与CD11b+巨噬细胞中叉头框P3-绿色荧光蛋白（Foxp3GFP）的表达水平（MM5数据集）。以CD4 T细胞的阴性荧光峰作为阈值划定阳性群边界，采用未携带GFP转基因的野生型小鼠作为染色实验的“真阴性”对照。
本数据集包含本次分析中使用的传统补偿算法与AutoSpill对应的荧光补偿矩阵。