Expression of stemness factors, such as octamer-binding transcription factor 3/4 (OCT3/4), sex determining region Y-box 2 (SOX2), and alkaline phosphatase (ALP) in human deciduous tooth-derived dental pulp cells (HDDPCs) can be assessed through fixation and subsequent immuno- or cytochemical staining. Fluorescence-activated cell sorting (FACS), a powerful system to collect cells of interest, is limited by the instrument cost and difficulty in handling. Magnetic-activated cell sorting is inexpensive compared to FACS, but is confined to cells with surface expression of the target molecule. In this study, a simple and inexpensive method was developed for the molecular analysis of immuno- or cytochemically stained cells with intracellular expression of a target molecule, through isolation of a few cells under a dissecting microscope using a mouthpiece-controlled micropipette. Two or more colored cells (~ 10), after staining with a chromogen such a 3,3′-diaminobenzidine, were successfully segregated from unstained cells. Expression of glyceraldehyde 3-phosphate dehydrogenase, a housekeeping gene, was discernible in all samples, while the expression of stemness genes (such as OCT3/4, SOX2, and ALP) was confined to positively stained cells. These findings indicate the fidelity of these approaches in profiling cells exhibiting cytoplasmic or nuclear localization of stemness-specific gene products at a small-scale.

中文翻译：

---

基于少量人工分离的固定细胞 (RNA-snMIFxC) 的 RNA 分析，以分析来自人类乳牙源牙髓细胞的干细胞

干性因子，如八聚体结合转录因子 3/4 (OCT3/4)、性别决定区 Y-box 2 (SOX2) 和碱性磷酸酶 (ALP) 在人乳牙源牙髓细胞 (HDDPC) 中的表达可以通过固定和随后的免疫或细胞化学染色进行评估。荧光激活细胞分选 (FACS) 是一种强大的收集目标细胞的系统，但受到仪器成本和操作难度的限制。与 FACS 相比，磁激活细胞分选是便宜的，但仅限于具有目标分子表面表达的细胞。在这项研究中，开发了一种简单且廉价的方法，用于对具有靶分子的细胞内表达的免疫或细胞化学染色细胞进行分子分析，通过使用咬嘴控制的微量移液管在解剖显微镜下分离一些细胞。两个或多个有色细胞 (~ 10)，在用 3,3'-二氨基联苯胺等色原染色后，成功地与未染色的细胞分离。3-磷酸​​甘油醛脱氢酶（一种管家基因）的表达在所有样品中均可见，而干细胞基因（如 OCT3/4、SOX2 和 ALP）的表达仅限于染色阳性的细胞。这些发现表明这些方法在分析细胞中的保真度，这些细胞在小范围内表现出干性特异性基因产物的细胞质或核定位。3-磷酸​​甘油醛脱氢酶（一种管家基因）的表达在所有样品中均可见，而干细胞基因（如 OCT3/4、SOX2 和 ALP）的表达仅限于染色阳性的细胞。这些发现表明这些方法在分析细胞中的保真度，这些细胞在小范围内表现出干性特异性基因产物的细胞质或核定位。3-磷酸​​甘油醛脱氢酶（一种管家基因）的表达在所有样品中均可见，而干细胞基因（如 OCT3/4、SOX2 和 ALP）的表达仅限于染色阳性的细胞。这些发现表明这些方法在分析细胞中的保真度，这些细胞在小范围内表现出干性特异性基因产物的细胞质或核定位。