Experiment 4: Chemotaxis Under Agarose

 

 

The purpose of this assay is to compare the speed and directionality of NC4A2 and HK321 cells during chemotaxis towards folate.  You can set up several variations on the basic experiment: different agarose concentrations, folate concentrations or use different numbers of cells in different plates.

 

For best results, change the media on your cells the day before the assay so they think they are in log phase.

 

Procedure

 

Pouring Agarose Plates

 

This assay uses SM media. We will discuss why we are using SM media in lecture. You want 8 ml (agar + SM) per P60 dish. You can use anywhere between 0.75% agarose (0.75g/100ml) and 2.5% agarose (2.5g/100ml). Plates may be prepared a day in advance.

 

1.  To prepare several dishes, combine the appropriate amount of agarose with SM media in a flask. 

 

2.  Melt the agarose/SM in the microwave.  Watch carefully and stop when it begins to boil.  It will easily boil over if you are not careful. Open the microwave door and gently swirl the flask.  Be careful as the superheated fluid can boil over when you do this.  Wear protective gloves.  Repeat the heating to boiling 3-4 times until the you cannot see any agarose floating in the flask.

 

3.  Pipet 8 ml of melted agarose into the p60 Petri dishes and allow to cool on a level surface (takes about 15-30 minutes to harden).

 

            Prepare the cells while the plates are cooling.

 

 

Preparing NC4A2 and HK321 cells for Chemotaxis

 

You should do the assay with cells in log phase of growth, but it will probably work to some extent no matter what state your cells are in.  For best results, change the media on your cells the day before the assay so they think they are in log phase.

 

You will need 100 μl of cells at about 106-107/ml for each trough. You will do two troughs per plate.  I would aim for the higher number since it is easier to see the results with more cells. So make sure you prepare more than more than 100 μl of cells in case you need more to fill the troughs. 

 

1.     Triturate your cells into a 15ml centrifuge tube, titer the cells while they are spinning down, and figure out what volume to resuspend in to get the right concentration.

 

2.    Spin down cells for 5 min at 1000 rpm at 4°C.

 

3.    Aspirate HL-5, vortex the tube and then add the proper volume of SM to bring concentration to ~107cells/ml and then vortex the cells.

 

 

Cutting the troughs in the agarose

 

1.    The troughs should be cut the length of a single edged razor blade, 2-5 mm wide and 5mm apart (See last page for a template). Blades can be reused, but you want it sharp to make a clean cut.

 

2.  Lay the plate on top of the template so you have a cutting guide.  Use your first plate as a trial plate.   Make several troughs in this plate to get a feel for the cutting action. The blade tends to cuts better after wetting by the agarose as well. Cut by laying the blade flat on the agarose surface and push down so the entire blade cuts through at once to the bottom.  Don’t try to saw your way through.  Try not to score the plastic as that will interfere with cell movement.

 

3.  After the troughs are cut, you can use a micropipet and yellow tip or a pointed spatula to lift the edge of the agar block and lift it out to form a trough. This is a non-sterile procedure, so you can use your fingers.

 

 

Adding cells and chemoattractant

 

1.     After vortexing your tube again, add about 100 μl of cells into each of your troughs. If the volume is not enough to fill the trough, add some more cells until the volume reaches up to the near the rim of the trough but don’t overfill. Add NC4A2 to one outside trough and HK321 cells to the other outside trough so you can compare the mutant and the wild type. 

 

2.    Now add the 0.1mM folic acid chemoattractant in the center trough.  Record the time, and begin preparing your cells while the gradient begins to diffuse.  Be Gentle handling the plates so you don’t spill cells or chemoattractant out of the troughs.

 

 The gradient will take about 1 hour to reach the cells and is still present up to 9 hours later.

 

 

 

Imaging 

 

1.    Within one to two hours, the first few cells start to exit the trough, and the plate may be imaged over the next 6-8 hours moving in the gradient.  You will get the best images if you wait until the cells are a field of view away from the trough.   The trough edges and the meniscus, interfere with the cone of light from the condenser.  If you want to image near the trough, gently put a coverslip over the agarose and then fill the trough so that the liquid reaches the coverslip.  This eliminates the meniscus effect.

 

2.    Record the movement of the cells over time. Focus on the cells farthest from the trough.  These leading cells will be more accurate in their gradient sensing because the cells behind them are seeing a gradient that has been disturbed by the cells in front of them.  You can measure the movement of both and should be able to show this directly.

 

3.    In addition to imaging at 10x for speed, you should image the cells at 40x so you can see the actual mechanism of movement.  It may look different from the vegetative cells you have imaged before. 

 

 

Analysis: Use ImageJ and mTrackJ plugin make measure the distance and speed of cells and make movies of cells with tracks. You should measure the parameters of movement (speed, persistence, directionality) and compare that to the vegetative cells and cAMP-induced aggregating cells you have previously measured.  You can also use the Set Reference feature of mTrackJ to tell the program where the folate source is and measure movement relative to that directional reference. 

  

For your Lab Report and Website

 

Present images, movies and tables to summarize the questions you asked and the answers you found.  How does this data compare to what you found with wild-type and mutant in vegetative movement.  How does it compare with movement in cAMP mediated movement during development?  If these are different, why do you think they are different?

 


 


















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Reagents/Media:

 

HL5 media

 

10 g BBL Thoitone E peptone

5 g yeast extract

10 g glucose

0.35 Na2HPO4

0.35 KH2PO4

Fill up to 1L with H2O

Adjust pH to 6.5-6.7

Autoclave

 

SM liquid media 

10 g Difco Bacto-Peptone

10 g glucose

1 g yeast extract

1.9 g KH2PO4

0.6 g K2HPO4

0.43 g MgSO4

Fill up to 1L with H20

Adjust pH to 6.5-6.7

Autoclave

 

Folate

Use 0.1mM Folic acid diluted from the stock solution into SM media

Stock solution: 50 mM in NaOH:

Rifkin recipe- Dissolve folic acid in 0.1N NaOH to 50 mM and store dark and cold.  This is a 500x stock for under agar.  Dilute in KK2 or SM or other buffer for use.  Make a fresh dilution approximately once a week.  According to Rifkin the stock is stable for at least 1 year at 4C in dark.

 

Cell Strain(s):

HK321

NC4A2

 

Other things needed for this experiment:

1.      p60 dishes

2.      razor blades

3.      agarose

4.      clean 125 ml flasks (for boiling agarose)

5.      microwave oven

 
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