CSF extract, mike's protocol (Stukenberg lab)

Protocol submitted by VGP from Stukenberg lab protocols [1]

link to protocol page [2]

Mike's CSF Extract Protocol

1. Five days prior to planned extract date, prime frogs with PMSG, 50 units/frog (this dramatically improves the amount of extract l).

2. Approx 16hrs before preparing extract, inject frogs with Human Chorianic Ganadotropin (HCG), 600 units/frog.

3. Place 4-6 liters of H2O in 20 °C incubator. 

4. If you will be collecting laid eggs place the frogs in buckets containing MMR.  If only using fresh eggs leave the frogs in water overnight.  Collecting only fresh or squeezed eggs improves egg quality but severely compromises the amount of eggs that will be collected. 

5. Come in to lab early, enjoy some fresh coffee and begin to set-up for making extracts.

Prepare:

· 2-3 liters of MMR: 5 mM Na-HEPES (pH 7.8), 0.1 mM EDTA, 100 mM NaCl, 2 mM KCl, 1 mM MgCl2, 2 mM CaCl2.  Prepare from a 10x  or 25x stock

· 750-1000ml XB: 10 mM K-Hepes (pH 7.7), 100mM KCl, 1 mM MgCl2, 0.1 mM CaCl2, 50 mM sucrose. Maintain pH of 7.7 w/ 11 ml of 10N KOH per 100 ml.  Make with 20x XB salts, 2 M sucrose, 1 M K-Hepes (pH 7.7)

· 250 ml CSF-XB: XB + 1 mM MgCl2 + 5mM EGTA.  Take 250 ml of XB that was just made and add MgCl2 and EGTA.

· 100 ml CSF-XB + Protease Inhibitors:  CSF-XB + 10 mg/ml LPC. Use 100 ml of CSF-XB just made.

· 200 ml de-jellying solution: 2 % (w/v) cysteine in 1x XB salts, pH to 7.8 with 0.9 ml of 10N NaOH.  Use 20x XB salts and cysteine.  DO NOT PREPARE CYSTEINE UNTIL IMMEDIATELY BEFORE IT IS NEEDED.

6. Collect eggs in fresh MMR; good CSF and cycling extracts depends on the quality of eggs collected.  Always take care to handle eggs and extract very gently.

7. Remove white puffy eggs, stringy eggs, eggs that are mis-shapen and those that generally don’t look bad.  It is generally better to compromise quality for quantity.

8. Wash eggs 2-3 in MMR.  Pour off as much MMR as possible.

9. Again, remove any puffballs and/or poor quality eggs.

10. Add de-jellying solution to eggs, intermittently swirling gently.  De-jellying takes 5-10 minutes.  After they are dejellyed, there volume will decrease up to five fold and they will pack much tighter. 

11. Pour off de-jellying solution.

12. Wash de-jellied eggs 2-4 times with XB.

13. Wash eggs 2-3 times with CSF-XB.

14. Wash eggs 2 times with CSF-XB + PI's.

15. Transfer eggs to a 2.2 ml thin, clear plastic centrifuge tube containing 2 ml of LPC and 10 ml of cytochalasin B.  The size of the tube depends on the number of eggs collect.  For lower numbers of eggs, use a smaller tube. 

16. Aspirate excess buffer off of the eggs.

17. Place in a round bottom culture tube and spin in a clinical centrifuge on low for 30 seconds.

18. Aspirate off as much buffer as possible and add 100 - 200 ml of versalube oil.

19. In clinical centrifuge spin for 30 seconds on low and 60 seconds on high. 

20. Aspirate ALL buffer and versalube from the top of packed eggs.

21. Transfer the tube to a HB-4 rotor and spin at 10,000 rpm, 15 minutes, 16 °C (or the equivalent in a similar spinning bucket rotor)

22. Store the crushed eggs on ice.  The top yellow layer is the lipid layer and the dark bottom layer is yolk and nuclei.  The middle layer is the desired cytoplasmic extract.

23. After wiping the outside of the tube with a alcohol wetted kimwipe, insert an 18-gauge needle into the side of the tube (BE CAREFUL), and draw out the cytoplasmic layer.  Use a new needle for each tube.

24. Place the fresh extract into a clean pre-chilled tube on ice.

25. Estimate the volume of extract obtained and add 10mg/ml LPC, cytochalasin, 1x final concentration energy mix, and 1/40 volume of 2 M sucrose.  Add into cap and then tip a couple of times to mix. 

26. Test to determine if the extract is arrested in mitosis.  To do so, take 15 mL of fresh extract and to a tube that contains 1 ml of diluted sperm for experiment.  Remove 7.5 ml and add to a tube containing 1 ml of 4 mM CaCl2.  Place at room temperature for 20-30 minutes. 

27. Place 1 ul of extract onto a plane glass slide and overlay with 3 ml of extract fix (100 ml 10x MMR, 0.1 ml Hoechst, 300 ml 37% formaldehyde, 600 ml of 80% Glycerol).  Examine under the microscope.  The sample containing calcium should exit mitosis and have interphase looking nuclei (rounded morphology). The sample without calcium should remain in mitosis and contain condensed mitotic chromatin.