Fluorescence-activated cell sorting (FACS) is a branch of flow cytometry that allows for the isolation of specific cell populations that can then be further analyzed by single-cell RNA sequencing (scRNA-seq). When utilizing FACS for population isolation prior to sequencing, it is essential to consider the protection of RNA from RNase activity, environmental conditions, and the sorting efficiency to ensure optimum sample quality. This study aimed to optimize a previously published MDSC flow cytometry strategy to FACS sort canine Myeloid-Derived Suppressor Cells (MDSC) with various permutations of RNAlater ™ and RiboLock™ before and after FACS sorting. Concentrations of RNAlater™ greater than 2 % applied before flow analysis affected cell survival and fluorescence, whereas concentrations ≤ 2 % and time ≤ 4 h had little to no effect on cells. To shorten the procedural time and to enhance the sorting of rare populations, we used a primary PE-conjugated CD11b antibody and magnetic column. The combination of RiboLock™ pre- and post-sorting for FACS provided the best quality RNA as determined by the RNA integrity number (RIN ≥ 7) for scRNA-seq in a normal and dog and a dog with untreated oral melanoma dog. As proof of principle, we sequenced two samples, one from a normal dog another from a dog with untreated oral melanoma. Applying scRNA-Seq analysis using the 10X Genomic platform, we identified 6 clusters in the Seurat paired analysis of MDSC sorted samples. Two clusters, with the majority of the cells coming from the melanoma sample, had genes that were upregulated (> log2); these included MMP9, MMP1, HPGD, CPA3, and GATA3 and CYBB, CSTB, COX2, ATP6, and COX 17 for cluster 5 and 6 respectively. All genes have known associations with MDSCs. Further characterization using pathway analysis tools was not attempted due to the lower number of cells sequenced in the normal sample. The benefit deriving from the results of the study helped to gain data consistency when working with cells prone to RNase activity, and the scRNA-seq provided data showing transcriptional heterogeneity in MDSC populations and potentially identifying previously unreported or rare cell populations.

荧光激活细胞分选（FACS）是流式细胞仪的一个分支，它可以分离特定的细胞群，然后可以通过单细胞RNA测序（scRNA-seq）对其进行进一步分析。在测序之前使用FACS进行群体分离时，必须考虑保护RNA免受RNase活性，环境条件和分选效率的影响，以确保最佳的样品质量。本研究旨在优化与RNA的各种置换以前发表的MDSC流式细胞术策略来FACS排序犬髓源抑制细胞（MDSC）后™和的RiboLock™之前和之后FACS分选。稍后RNA浓度™在流动分析之前应用大于2％的™会影响细胞存活和荧光，而浓度≤2％和时间≤4 h对细胞几乎没有影响。为了缩短程序时间并增强稀有种群的分类，我们使用了与PE偶联的主要CD11b抗体和磁柱。RiboLock™预分选和后分选的FACS组合可提供最佳质量的RNA，这是由正常人和狗以及未经治疗的口腔黑素瘤狗的狗中scRNA-seq的RNA完整性数（RIN≥7）确定的。作为原理证明，我们对两个样品进行了测序，一个样品来自正常狗，另一个样品来自未经治疗的口腔黑色素瘤的狗。应用使用10X Genomic平台的scRNA-Seq分析，我们在MDSC分选样品的Seurat配对分析中确定了6个簇。两个集群 大多数细胞来自黑色素瘤样本，其基因被上调（> log2）；这些包括MMP9，MMP1，HPGD，CPA3和GATA3以及CYBB，CSTB，COX2，ATP6和COX 17分别适用于群集5和6。所有基因都具有与MDSC的已知关联。由于正常样品中测序的细胞数量较少，因此未尝试使用途径分析工具进行进一步表征。从研究结果中获得的好处有助于在处理易于产生RNase活性的细胞时获得数据一致性，而scRNA-seq提供的数据显示了MDSC群体中的转录异质性，并有可能鉴定出以前未报道或罕见的细胞群体。