Products Description
Proteolytic enzymes are widely used in cell dissociation. With some tissues papain has proved less damaging and more effective than other proteases. Lam found that of the enzymes used for dissociating turtle retina, papain produced the least trauma. Intact single photoreceptor cells have been isolated from adult salamander retina with papain. Huettner and Baughman described a method using papain to obtain high yields of viable, morphologically intact cortical neurons from postnatal rats. Finkbeiner and Stevens applied the Huettner and Baughman method to the dissociation of postnatal rat hippocampus. Papain is used with fetal as well as postnatal brain regions to provide maximal dissociation and viability of neurons.
The Worthington Papain Dissociation System is a set of reagents intended for use in the tissue dissociation method of Huettner and Baughman. The materials are designed for convenience and simplicity and are useful to the occasional user as well as the more experienced and frequent user. Each lot is use tested for performance in tissue dissociation and provides freshly prepared enzyme solutions for each dissociation.
The reagents are stable at ambient temperatures for the periods of time expected in normal shipping procedures, but the package should be refrigerated upon arrival and can be stored at 4-8°C for up to four months before use.
Information
Description and Package Contents:
The package contains sufficient materials for dissociation of five separate tissue aliquots of up to 0.3 - 0.4 cm3 each. For larger tissue samples prepare proportionately larger volumes of reagents at each step and combine them in the same ratio as described in the protocol.
- Vial 1
- Sterile Earle's Balanced Salt Solution (EBSS) with bicarbonate and phenol red, one vial per package.
- Aliquots of this vial are used to reconstitute other vials and to prepare dilute inhibitor solution. Refrigerate between uses and equilibrate with sterile O2:CO2 before each use.
- Vial 2
- Papain containing L-cysteine and EDTA, five single use vials per package.
- This material is 0.22 micron membrane filtered and lyophilized in autoclaved vials. A vial reconstituted with five mls of EBSS (vial 1) yields a solution at 20 units of papain per ml in one millimolar L-cysteine with 0.5 millimolar EDTA. Brief incubation is needed to insure full solubility and activity.
- Vial 3
- Deoxyribonuclease I (DNase), five single use vials per package.
- This material is 0.22 micron membrane filtered and lyophilized in autoclaved vials. A vial reconstituted with 0.5 ml of EBSS (vial 1) yields a solution at 2000 units of deoxyribonuclease per ml. Avoid vigorous mixing.
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- Vial 4
- Ovomucoid protease inhibitor with bovine serum albumin, one vial per package.
- This material is 0.22 micron membrane filtered and lyophilized in autoclaved vials. A vial reconstituted with 32 mls of EBSS (vial 1) yields a solution at an effective concentration of 10 mgs of ovomucoid inhibitor and 10 mgs of albumin per ml. The inner rubber stopper can be discarded after reconstitution. Aliquots of this vial are used for each dissociation. Refrigerate between uses and equilibrate with sterile O2:CO2 before each use. Stable after reconstitution when stored at 4°C.
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- Also included is a card correlating color with pH for use as a guide in O2:CO2 equilibration.
Needed but not supplied:
- Sterile pipets
- Sterile centrifuge tubes
- Centrifuge to operate at 70g and 300g
- Device for sterile 95% O2:5%CO2 equilibration of solutions
- Water bath at 37°C
- Device for incubation at 37°C with agitation.
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