Lamina Propria Mononuclear Cell Isolation: A Silica-based Density Gradient Centrifugation Technique to Obtain Mononuclear Cells from Murine Colon
Transcription
The intestinal lamina propria, located below the intestinal epithelium, consists of loose connective tissue along with a dense network of collagen fibers containing a diverse array of cells such as fibroblasts, mononuclear cells, including lymphocytes and monocytes. These cells play a crucial role in maintaining homeostasis.
To isolate mononuclear cells, begin by taking mouse colon fragments. Treat the fragments with a digestion buffer containing collagenase enzymes. Collagenase enzymes digest the collagen fibers within the tissue, releasing the cells into suspension.
Collect the supernatant and filter it through cell strainers to remove undigested tissue and debris and obtain a single-cell suspension. Add a chilled buffer to the filtrate to inactivate the collagenase enzymes.
Centrifuge to separate the cells. Discard the supernatant containing the enzymes and resuspend the cell pellet in a low-density silica-based separation media. Next, layer the suspension over a high-density silica-based separation media to create a separation gradient.
Centrifuge the mixture. The mononuclear cells sediment and form a white layer at the interface between the density gradient layers. Carefully transfer the white interface layer into a fresh tube containing a suitable buffer.
Centrifuge to obtain a pellet containing purified mononuclear cells. Resuspend the pellet in buffer for further downstream analysis.
