Retro-orbital CD45 antibody labeling to evaluate antibody-secreting cell trafficking in mice.

Antibody-secreting cells (ASCs) are critical regulators of the humoral immune response. However, differences between tissue resident populations versus those that have recently migrated to their final anatomic destination are poorly understood. Here, we present a protocol for using retro-orbital (r.o.) CD45 antibody labeling to identify tissue resident versus recently immigrated ASCs in mice. We describe steps for r.o. injection of antibodies, animal euthanasia, and tissue harvesting. We then detail tissue processing, cell counting, and cell staining for flow cytometry analysis. For complete details on the use and execution of this protocol, please refer to Pioli et al. (2023). Protocol for r.o. CD45-PE antibody labeling to measure ASC trafficking Steps to harvest and process tissues for cell staining Steps for surface staining of cells preceding flow cytometric analysis Examples of how to analyze and interpret flow cytometry data Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Antibody-secreting cells (ASCs) are critical regulators of the humoral immune response. However, differences between tissue resident populations versus those that have recently migrated to their final anatomic destination are poorly understood. Here, we present a protocol for using retro-orbital (r.o.) CD45 antibody labeling to identify tissue resident versus recently immigrated ASCs in mice. We describe steps for r.o. injection of antibodies, animal euthanasia, and tissue harvesting. We then detail tissue processing, cell counting, and cell staining for flow cytometry analysis.

抗体分泌细胞（ASCs）是体液免疫反应的关键调节因子。然而，组织驻留群体与那些近期迁移到其最终解剖位置的群体之间的差异却鲜为人知。在此，我们介绍一种使用眼眶后（r.o.）CD45抗体标记来识别小鼠中组织驻留的与近期迁入的ASCs的实验方案。我们描述了眼眶后注射抗体、动物安乐死以及组织采集的步骤。然后我们详细阐述了用于流式细胞术分析的组织处理、细胞计数和细胞染色。 关于该方案的使用和实施的完整细节，请参考皮奥利等人（2023年）的研究。 眼眶后CD45 - PE抗体标记以测量ASC转运的方案 用于细胞染色的组织采集和处理步骤 在流式细胞术分析之前细胞表面染色的步骤 如何分析和解释流式细胞术数据的示例 出版商注：进行任何实验方案都需要遵守当地机构关于实验室安全和伦理的指导方针。 抗体分泌细胞（ASCs）是体液免疫反应的关键调节因子。然而，组织驻留群体与那些近期迁移到其最终解剖位置的群体之间的差异却鲜为人知。在此，我们介绍一种使用眼眶后（r.o.）CD45抗体标记来识别小鼠中组织驻留的与近期迁入的ASCs的实验方案。我们描述了眼眶后注射抗体、动物安乐死以及组织采集的步骤。然后我们详细阐述了用于流式细胞术分析的组织处理、细胞计数和细胞染色。