Preparation of Tissue Samples for Cell Separation
When isolating cells from intact tissues, you must first disrupt the extracellular matrix holding the cells together using mechanical force and/or proteolytic enzymes. Creating a single-cell suspension is particularly important when using cell separation techniques that rely on cell labeling, as incomplete dissociation of cell clumps can lead to inefficient labeling of the target cells. It is also important to ensure that the processing method does not affect cell surface epitopes, as this may negatively impact both cell isolation and downstream functional analyses.
There are several enzymes commonly used in tissue dissociation protocols:
- Collagenase can hydrolyze collagen and is widely used for isolating cells from animal tissues.
- Hyaluronidase is often used in combination with collagenase and catalyzes hydrolysis of 1,4-β-D-glycosidic linkages.
- DNase is added to cell suspensions to minimize cell clumping due to DNA released by damaged cells.
- Elastase is used to digest tissues containing high amounts of elastin.
- Trypsin is a serine protease with a specificity for peptide bonds and is often combined with other enzymes (e.g. elastase and/or collagenase) for tissue dissociation.
Tissue dissociation conditions (e.g. enzyme concentration, type of enzyme, and incubation time) must be optimized in order to maximize cell yield and viability.
Even with careful optimization, tissue preparation protocols can result in some cell death. To ensure that apoptotic cells do not interfere with your experiments, they can be depleted from your sample (e.g. with the EasySep™ Dead Cell Removal (Annexin V) Kit) prior to performing further cell isolation or downstream assays.
Prepare Lymphoid Tissues for Cell Isolation
Spleens are lymphoid tissues that are rich sources of immune cells. In order to isolate immune cell subsets from lymphoid tissues, you can easily prepare single-cell suspensions of splenocytes using mechanical force. Watch this video to learn about this common way to prepare mouse splenocytes.
Preparing single-cell suspensions of other lymphoid tissues, such as lymph nodes, can be made by following a similar protocol.
Prepare Non-Lymphoid Tissues for Cell Isolation
Immune cells isolated from non-lymphoid tissues are commonly used to study the phenotype and function of tissue-resident immune cells or to investigate immune responses at specific disease sites. Methods for preparing single-cell suspensions from non-lymphoid tissue samples can vary greatly across tissue and final desired cell type. Our product information sheets outline some common tissue dissociation protocols to prepare non-lymphoid tissues, including central nervous system, intestinal, and lung tissues.
Tissue Dissociation Protocols
Cell Separation Methods and Techniques
Use one of these techniques to isolate cells from your tissue sample.
Overview of Cell Separation
Learn everything you need to know about cell separation.