Dissociating cells from specific tissue types requires specific parameters for tissue agitation to obtain a high volume of viable, culturable cells. The Miltenyi gentleMACS dissociator optimizes this task with a simple, practical protocol. In this publication the use of this apparatus on lung tissue is explained.
To work under sterile conditions, it is recommended to perform all steps in a laminar flow hood.
1. Materials
2. Dissociating the lung tissue
3. Filtration
Part 4: Representative Results:
Figure 1. Lung dissociation with the gentleMACS™ Dissociator resulted in 92% viable cells. Dead cells were fluorescently stained with propidium iodide (PI) ( right dot plot; left dot plot: no PI staining).
Figure 2. Dissociation of mouse lung tissue using the gentleMACS Dissociator results in a high percentage of viable leukocytes and endothelial cells. The derived single-cell suspensions were stained with CD45-PE and CD146-FITC or CD31-FITC and analyzed by flow cytometry.
Figure 3. Single-cell suspension derived from mouse lung tissue was stained with CD11c-FITC and Anti-mPDCA-1-APC to detect mouse CD11clow m-PDCA-1+ plasmacytoid DCs as well as CD11chigh cells.
Figure 4. Enrichment of dendritic cells using CD11c MicroBeads, a MiniMACS Separator and two MS Columns.
In this video, we introduce a new method for the dissociation of mouse lung tissue. We show that a combination of mechanical and enzymatic treatment of lung tissue yielded a high percentage of viable leukocytes and endothelial cells. Specifically, the mechanical disintegration of the tissue was achieved by the gentleMACS Dissociator. The gentleMACS C Tubes include a rotor – stator system, which dissociates tissue in a gentle way. The procedure is controlled by the program settings of the instrument. The settings were optimized to achieve high yield and viability of cells. The derived single-cell suspensions were stained with CD45-PE and CD146-FITC or CD31-FITC and analyzed by flow cytometry to detect leukocytes and endothelial cells. Dendritic cells in the single-cell suspension were stained with CD11c-FITC and Anti-mPDCA-1-APC. Furthermore, dendritic cells from a mouse lung single-cell suspension were enriched using MACS Technology: dendritic cells were magnetically labeled using CD11c MicroBeads and separated using a MiniMACS Separator and MS Columns. 1,2 In general, the combination of our new dissociation protocol with magnetic cell sorting represents a standardized method to obtain specific cell types from lung tissue.
The authors have nothing to disclose.
The authors are employees of Miltenyi Biotec GmbH, Germany.