Guidelines for DC preparation and flow cytometry analysis of mouse lymphohematopoietic tissues

Abstract

This article is part of the Dendritic Cell Guidelines article series, which provides a collection of state‐of‐the‐art protocols for the preparation, phenotype analysis by flow cytometry, generation, fluorescence microscopy, and functional characterization of mouse and human DC from lymphoid organs, and various non‐lymphoid tissues. Within this chapter, detailed protocols are presented that allow for the generation of single‐cell suspensions from mouse lymphohematopoietic tissues including spleen, peripheral lymph nodes, and thymus, with a focus on the subsequent analysis of DC by flow cytometry. However, prepared single‐cell suspensions can be subjected to other applications including sorting and cellular enrichment procedures, RNA sequencing, Western blotting, and many more. While all protocols were written by experienced scientists who routinely use them in their work, this article was also peer‐reviewed by leading experts and approved by all co‐authors, making it an essential resource for basic and clinical DC immunologists.

Guidelines for mouse and human DC functional assays

“…2). The identification of the DC subsets was performed as described in "Guidelines for DC preparation and flow cytometry analysis of mouse lymphohematopoietic tissues" [9]. FlowJo v10.8 (BD) was used to extract the mean fluorescence intensity values for cDC1 and cDC2 DC.…”

“…66. Prepare splenic single-cell suspensions (or single-cell suspension from your organ of interest) by digestion with Collagenase D and DNAse I as described in "Guidelines for DC preparation and flow cytometry analysis of mouse lymphohematopoietic tissues" [9] containing protocols for the generation of DC containing single-cell suspensions from spleen, lymph nodes, and thymus. 67.…”

“… Prepare splenic single‐cell suspensions (or single‐cell suspension from your organ of interest) by digestion with Collagenase D and DNAse I as described in “Guidelines for DC preparation and flow cytometry analysis of mouse lymphohematopoietic tissues” [9] containing protocols for the generation of DC containing single‐cell suspensions from spleen, lymph nodes, and thymus. Re‐suspend the cells in the appropriate volume of RPMI+5%FCS medium (e.g., if the internalization into 5×10 6 cells in 50 μl is planned, re‐suspend the cells in a concentration of 200×10 6 cells/ml to seed 25 μl). …”

“…">Wash the cells three times with FACS buffer as for a usual staining. Optional: Stain the single‐cell suspension with additional antibodies to identify your populations of interest (e.g., DC subsets, see “ Guidelines for DC preparation and flow cytometry analysis of mouse lymphohematopoietic tissues” [9]. …”

“… Prepare splenic single‐cell suspensions (or single‐cell suspension from your organ of interest) by digestion with Collagenase D and DNAse I as described in “ Guidelines for DC preparation and flow cytometry analysis of mouse lymphohematopoietic tissues” [9]. …”