Creatine Kinase Assay
Materials and Methods
0.5% Tetracain Hydrochloride (Bausch and Lomb # 24208-020-64)Microhematocrit tubes, 75mm heparinized (Fisherbrand # 22-362-566)
1.5 ml microcentrifuge tubes
Beckman Synchron CX5 Delta
Synchron Enzyme Validator Set (Beckman #44135)
Creatine Kinase Kit (Beckman #467830)
Peri-orbital Bleeding:
1) The week before peri-orbital bleeding, mice to be tested should be kept in a quiet place. During this time, the bedding should not be changed and the animals should be disturbed as little as possible.
2) ENU mutagenized G3 hybrid mice (C57BL/6J and BALB/cByJ mix) are tested at 3 and 6 months of age by peri-orbital bleeding. The peri-orbital venous sinus of the mouse may be used as a source of venous blood, which can be collected from either the medial or lateral canthus.
3) Using the standard peri-orbital bleeding protocol and following the application of a topical anesthetic (tetracain hydrochloride), obtain approximately two microhematocrit tubes of blood. Eye bleeding is best performed in the middle of the sleep cycle, approximately at noon. Since activity can cause increased total Creatine Kinase (CK) levels, the activity of the mouse at the time of blood collection should be noted. Scores of 1-5 are given.
Removal of Plasma and Storage:
4) Transfer the blood sample into a pre-numbered 1.5 ml microcentrifuge tube. It is important to handle the samples very carefully to avoid hemolysis (the lysing of the red blood cells [RBCs]).
5) Spin the samples at 5000 rpm (2500xg) for 5 minutes. Do not spin longer or at higher speeds as hemolysis may occur. Immediately remove the plasma from the top of the tube, avoiding the RBCs. A maximum allowable time between peri- orbital bleeding and removal of the plasma is two hours.
Note: Samples must be assayed within 4 hours of blood collection if they remain at room temperature or may be stored at 4oC and assayed within 12 hours. Alternatively they may be stored at -70oC for up to 6 weeks. Do not place samples on ice, as this can lead to increased hemolysis.
6) Repeat step 5 to remove any remaining RBCs and transfer the plasma to a new tube. Note the color of the plasma and give it a code from 1-5. It should have a golden appearance. The more reddish it appears the more hemolysis has occurred. Store the plasma at -70oC.
Analysis of Sample:
7) Thaw the sample immediately before use. Do not freeze/thaw.
8) Transfer 60µl into a 0.5 ml sample cup and load onto a Beckman Synchron CX5, Delta that has been precalibrated for total CK. Samples may be diluted with a buffered saline solution containing 1% BSA.
9) Normal total CK ranges for a mouse are between 35- 200 IU/L. Values over 500 IU/L are retested. Mice with values that remain over 500 IU/L and have low scores for both color and activity are flagged as deviant and selectively bred to determine potential heritability.
