PhyMedExp, University of Montpellier, INSERM U1046, CNRS UMR9214, France; Medicine Biology Mediterranee, Department of Respiratory Diseases and Addictology, Arnaud de Villeneuve Hospital, CHRU Montpellier, France.
Medicine Biology Mediterranee, Department of Respiratory Diseases and Addictology, Arnaud de Villeneuve Hospital, CHRU Montpellier, France.
Biochem Biophys Res Commun. 2022 May 14;604:151-157. doi: 10.1016/j.bbrc.2022.03.040. Epub 2022 Mar 12.
As opposed to surface marker staining, certain cell types can only be recognized by intracellular markers. Intracellular staining for use in cell sorting remains challenging. Fixation and permeabilization steps for intracellular staining and the presence of RNases notably affect preservation of high-quality mRNA. We report the work required for the optimization of a successful protocol for microarray analysis of intracellular target-sorted, formalin-fixed human bronchial club cells. Cells obtained from differentiated air-liquid interface cultures were stained with the most characteristic intracellular markers for club cell (SCGB1A1) sorting. A benchmarked intracellular staining protocol was carried out before flow cytometry. The primary outcome was the extraction of RNA sufficient quality for microarray analysis as assessed by Bioanalyzer System. Fixation with 4% paraformaldehyde coupled with 0.1% Triton/0.1% saponin permeabilization obtained optimal results for SCGB1A1 staining. Addition of RNase inhibitors throughout the protocol and within the appropriate RNA extraction kit (Formalin-Fixed-Paraffin-Embedded) dramatically improved RNA quality, resulting in samples eligible for microarray analysis. The protocol resulted in successful cell sorting according to specific club cell intracellular marker without using cell surface marker. The protocol also preserved RNA of sufficient quality for subsequent microarray transcriptomic analysis, and we were able to generate transcriptomic signature of club cells.
与表面标志物染色相反，某些细胞类型只能通过细胞内标志物来识别。用于细胞分选的细胞内染色仍然具有挑战性。固定和透化步骤用于细胞内染色，以及存在 RNase 会显著影响高质量 mRNA 的保存。我们报告了优化成功的细胞内目标分选、福尔马林固定人支气管 club 细胞的微阵列分析方案所需的工作。从分化的气液界面培养物中获得的细胞用最具特征性的 club 细胞（SCGB1A1）分选的细胞内标志物进行染色。在进行流式细胞术之前，进行了基准化的细胞内染色方案。主要结果是通过 Bioanalyzer 系统评估，提取足够质量用于微阵列分析的 RNA。用 4%多聚甲醛固定并结合 0.1% Triton/0.1%皂素透化，可获得最佳的 SCGB1A1 染色效果。在整个方案中添加 RNase 抑制剂，并在适当的 RNA 提取试剂盒（福尔马林固定石蜡包埋）中添加，可显著改善 RNA 质量，使样品有资格进行微阵列分析。该方案根据特定的 club 细胞细胞内标志物成功地进行了细胞分选，而无需使用细胞表面标志物。该方案还保存了足够质量的 RNA，用于随后的微阵列转录组分析，并且我们能够生成 club 细胞的转录组特征。
