Tissue culture of Xenopus cell lines S3, XTC (Stukenberg lab) and Assaying H1 kinase in extracts (Stukenberg lab): Difference between pages

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Tissue Culture Care Instructions for Xenopus cell linesS3, XTC
Histone H1 Kinase Assay from Xenopus Extracts
 
 
PurposeTest whether the H1 kinase Cdc2-CcyclinB is still active in an extract.  This is a readout for the extracts mitotic state.  If activity is high, the extract is mitotic, if it is low, the extract has exited mitosis and is in interphase.  These are normally done in our lab as a readout for the spindle checkpoint.  An extract containing sufficient sperm and nocodazole will maintain a mitotic state after being released from CSF, by the addition of calcium.
Background:  Snap freeze in liquid nitrogen 1µl samples of the extract to be analyzed.  Store at -80°C until you are ready to perform the kinase assay.
   
   
Media:  L-15 Medium (Leibovitz), Sigma-Aldrich product # L4386.  This comes in powder form, which we resuspend to a 66% solution (dissolve one 14.7g/L vial into 1.4L ddH2O and pH to 7.6).
Prepare a mixture of the following reagents, making enough for all kinase assays to be performed at one time.
 
New media must be filter sterilized into sterile bottles. We usually use Nalgene 500mL receiver bottles and  Store media at 4˚C. 
Before use with cells, you must add 10% FBS (fetal bovine serum),  1X Penn/Strep (antibiotic, comes as 100X stock), and 1X Sodium Pyruvate (also a 100X stock). 
Notes: Antibiotics are good for about 1 month, so if you’re using your media for longer than that, re-supplement it accordingly.
   
   
Getting started:


Keep everything sterile. Don’t use non-autoclaved tips, coverslips, or pipetsMake sure all dishes are sterile.
5x H1 Kinase Buffer - Amount per reaction: 2µl Final Concentration: 80mM b-Glycerophosphate, 12mM MgCl2, 15mM EGTA pH 7.4
Warm the media and trypsin (if using) to room temperature(I do this 30-60 minutes before I have hood time).
 
Make sure the UV light has been on in the hood you’re about to useBefore use, turn off the UV and turn on the blower. Spray gloved hands with 70% EtOHWipe down the surface of the hood with 70% EtOHSpray down and wipe all bottles, (basically anything going into the hood) with 70% EtOH.
ATP (10mM Stock) - Amount per reaction: 0.1 µl Final Concentration:100 µM
 
g32-P ATP - Amount per reaction: 0.1 µl
 
Histone H1 (10mg/ml Stock) - Amount per reaction: 0.125 µl Final Concentration: 1.25 µg
 
H2O 7.675 µl  
Add LPC to 10µg/ml, DTT to 1mM and NP-40 Substitute to 0.1%.  I usually mix NP-40 and water first and vortex into solution.   
5x H1 Kinase Buffer is stored in aliquots at -20°C.  
   
   
Splitting/Passaging Cells:
Histone H1 is made up in water and stored at -20°C
 
Remove media. Wash cells with 3-5mL of sterile Dulbecco’s Phosphate Buffered Saline (PBS).  Remove PBS and add 2mL 1XTrypsin (red color) per 100mm plate.  Swirl around and tap plates a few times.  Let sit a few minutes.  Add appropriate amount of fresh complete media to new plates for the dilution you’ve chosen.  S3 cells grow slowly, so don’t split them too dilute if you need to use them that same week.
Keep the mixture on ice until a few minutes before beginning the experimentAt this point place at room temperature for about 5 minutes, allowing it to warm up.
   
   
Growing cells on coverslips
Extract samples are moved from the -80°C freezer into an ice bucket immediately before beginning the experimentReactions are started by the addition of 9µl of the H1 Kinase mixture (above). Pipette up and down to mix the kinase assay solution and the extract, and incubate at room temperature for 30 minutesThe reactions are stopped by the addition of 20µl of 2x Laemmli Sample BufferSamples are run on SDS-PAGE gels, coomassie stained, destained, dried, and placed on a phosphoimager.   
 
Coverslips should be at the very least autoclaved or baked at 250˚C.  You can also HCl-wash and poly-lysine coat coverslipsCells are often happier on poly-lysine coated coverslips, so if you’re not happy with how the cells look, you may want to try these.   You can transfer coverslips to tissue culture dishes in two ways, either by tweezer (make sure it’s been cleaned well with EtOH, or by using the suction on a sterile Pasteur pipette (of course if your coverslips are in solution, this won’t work)Rinse coverslips a few times in PBS, and then add an appropriate volume of fresh media to themThen, split your cells as normal, adding a sufficient density of cells to each wellI’d advise splitting them pretty densely so that you can use them the next day if possible.  Lab lore indicates that the cells are more mitotic if you use them the next day.
   
   
Finishing up
  1.   Chen, R.H. and A.Murray. 1997. Characterization of spindle assembly checkpoint in Xenopus egg extracts. Methods Enzymol. 283:572-584.
 
Remove all your materials from the hood. Make sure you’ve turned off the aspirator, thrown away all pipettes, and plugged back in the pipetaid (if necessary). Wipe down the hood with 70% EtOH. Shut the hood, and turn on the UV light.

Latest revision as of 13:53, 15 November 2011

Protocol submitted by VGP from Stukenberg lab protocols [1]


link to protocol page [2]


Histone H1 Kinase Assay from Xenopus Extracts


Purpose: Test whether the H1 kinase Cdc2-CcyclinB is still active in an extract. This is a readout for the extracts mitotic state. If activity is high, the extract is mitotic, if it is low, the extract has exited mitosis and is in interphase. These are normally done in our lab as a readout for the spindle checkpoint. An extract containing sufficient sperm and nocodazole will maintain a mitotic state after being released from CSF, by the addition of calcium.

Background: Snap freeze in liquid nitrogen 1µl samples of the extract to be analyzed. Store at -80°C until you are ready to perform the kinase assay.

Prepare a mixture of the following reagents, making enough for all kinase assays to be performed at one time.


5x H1 Kinase Buffer - Amount per reaction: 2µl Final Concentration: 80mM b-Glycerophosphate, 12mM MgCl2, 15mM EGTA pH 7.4

ATP (10mM Stock) - Amount per reaction: 0.1 µl Final Concentration:100 µM

g32-P ATP - Amount per reaction: 0.1 µl

Histone H1 (10mg/ml Stock) - Amount per reaction: 0.125 µl Final Concentration: 1.25 µg

H2O 7.675 µl


Add LPC to 10µg/ml, DTT to 1mM and NP-40 Substitute to 0.1%. I usually mix NP-40 and water first and vortex into solution.

5x H1 Kinase Buffer is stored in aliquots at -20°C.

Histone H1 is made up in water and stored at -20°C

Keep the mixture on ice until a few minutes before beginning the experiment. At this point place at room temperature for about 5 minutes, allowing it to warm up.

Extract samples are moved from the -80°C freezer into an ice bucket immediately before beginning the experiment. Reactions are started by the addition of 9µl of the H1 Kinase mixture (above). Pipette up and down to mix the kinase assay solution and the extract, and incubate at room temperature for 30 minutes. The reactions are stopped by the addition of 20µl of 2x Laemmli Sample Buffer. Samples are run on SDS-PAGE gels, coomassie stained, destained, dried, and placed on a phosphoimager.

  1.   Chen, R.H. and A.Murray. 1997. Characterization of spindle assembly checkpoint in Xenopus egg extracts. Methods Enzymol. 283:572-584.