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Team:Cambridge/Protocols/Trypsin
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:# Samples of tissue were incubated in Hank's Buffered Saline Solution (HBSS) with a 2.5% trypsin solution for 30 minutes with gentle agitation of the tissue. | :# Samples of tissue were incubated in Hank's Buffered Saline Solution (HBSS) with a 2.5% trypsin solution for 30 minutes with gentle agitation of the tissue. | ||
| - | :# The supernatants were then gently removed by pipette and transferred to a clean centrifuge tube. Dispersed cells | + | :# The supernatants were then gently removed by pipette and transferred to a clean centrifuge tube. Dispersed cells were pelleted at 3000g for 10 minutes; the supernatant was discarded. |
| - | were pelleted at 3000g for 10 minutes; the supernatant was discarded. | + | |
:# The pelleted cells were resuspended in a little HBSS, sandwiched between a slide and coverslip then viewed. | :# The pelleted cells were resuspended in a little HBSS, sandwiched between a slide and coverslip then viewed. | ||
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===Safety=== | ===Safety=== | ||
Revision as of 14:34, 19 September 2011
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Trypsinisation
A method of enzymatically dispersing cells from tissue, for microscopy
Theory
Trypsin is a protease isolated from animal pancreas. It is one of the least-specific of the serine proteases so digests proteins thoroughly. When applied to tissue it digests the proteinaceous component of the extracellular space, the intracellular space remaining safe within the cell membrane. Application thus frees cells from the extracellular matrix, aiding their viewing with some types of microscopy.
Practice
- Samples of tissue were incubated in Hank's Buffered Saline Solution (HBSS) with a 2.5% trypsin solution for 30 minutes with gentle agitation of the tissue.
- The supernatants were then gently removed by pipette and transferred to a clean centrifuge tube. Dispersed cells were pelleted at 3000g for 10 minutes; the supernatant was discarded.
- The pelleted cells were resuspended in a little HBSS, sandwiched between a slide and coverslip then viewed.
Safety
The MSDS data sheet for trypsin states that it is harmful if swallowed, may act as a sensitiser by skin contact and is a skin, eye and respiratory irritant. Appropriate action should thus be taken.
