Spermatozoa Chemotaxis assay using Zigmond Chamber.

Friday, December 19, 2003

Description
This assay uses the zigmond chamber to study chemotaxis of spermatozoa or other migratory cells. The chamber consists of two wells separated by a bridge. The culture medium is placed on the bridge, cover with a coverslip and seal with parafin. The solution with the chemoattractant is placed in one well and the spermatozoa in the other. Cells will migrate toward the chemoattractant solution through the bridge. If there is chemotactic activity the spermatozoa will move toward the chemoattractant well if there is no activity they will move randomly over the bridge. Chemotaxis is expressed as a relation between the movement in the "x" axis over the "y" axis, where the "x" axis is in the direction of the chemoattractant gradient.

Procedure
Chamber Preparation

1. The chamber must be very well clean. Use a Zigmond chamber with a bridge of 1 or 2mm, preferently 1mm made of acrylic.
2. Put 10 ul of the medium where you culture the spermatozoa over the bridge.
3. Surrounding the wells and the bridge spread a thin layer of mineral oil.
4. Cover with a coverslip, and seal the borders with parafin.

B. Cell Preparation

1. The spermatozoa must be capacitated. Depending the species you work, you should use a capitation medium specific for your specie.
2. The concentration, also depends on the specie but should not be less than 3millons/ml.

C. Preparation of Chemoatractant Solutions

1. Dilute the chemoattractants to the desired concentrations in the same medium were you culture the spermatozoa. A negative control , must be included, spermatozoa culture medium with no chemoattractant. And a possitive control also must be included, generally could be, for mammalian spermatozoa, folicular fluid.

D. Assay Set Up

1. Fill one well with the chemoatractant solutions and seal the holes that you use for filled with parafin.
2. Fill the other well with the spermatozoa solution.
3. Keep the chamber at 37篊 for 10 min. to allow the spermatozoa to disperse, and also to allow the gradient of chemoatractant to be formed.
4. Then film for 5 min. the spermatozoa in the middle of the bridge, using a contrast phase microscopy and recorded it.
5. To analize chemotaxis, you should used an automatizated analizer like CASA or similar, or perform a semi-automatizared analysis, picking up the tracks manually and analyzing them with an appropriate software like Sigma ScanPro (SPSS) with or without SpermTrack (FCEFyN, UNC).

Recipes

Supplies

Tips
1. When you put the coverslip you should push it a little to avoid to have several focal fields.
2. The concentrations and the waiting time should be determined for your particular conditions and the specie that you work.
3. the coverslip and the chamber must be very clean, to avoid that the cells could be fixed to them.

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Spermatozoa Chemotaxis assay using Zigmond Chamber.