Mechanische dissociatie: een methode om levensvatbare cellen uit een weefsel te verkrijgen

First, weigh a tissue fragment and measure its length, breadth, and height. Place the tissue in a culture dish with a culture medium and dice it to small pieces using a scalpel. Transfer everything to a C-tube for homogenization using a Pasteur pipette.

Place the tube in a mechanical dissociator and run cycles as required. The apparatus uses mechanical forces to extract viable single cells from the tissue sample while maintaining cellular integrity, including the surface proteins. Decant the homogenate through a cell strainer fixed on a centrifuge tube.

Rehomogenize the remaining tissue and strain the homogenate through the cell strainer into the tube. Centrifuge the homogenate and remove the supernatant. Now, resuspend the cell pellet in the culture medium and transfer a small amount of the cell suspension to a tube containing trypan blue stain.

After staining, transfer the cells onto a hemocytometer slide. Count the transparent viable cells. The dead cells take up the stain, as their cell membrane is not intact. In the following protocol, we will perform non-enzymatic dissociation of fresh human tissue for quantitative and qualitative analysis of CD45+ cells.

- Begin by weighing