Multichannel-systems Roboinject Manual User Manual download pdf (Page 82)

Roboinject Manual

Barth’s solution without Ca

 pH 7.4 (with NaOH)

 88 mM NaCl

 2.4 mM NaHCO

 1 mM KCl

 0.82 mM MgSO

* 7 H

 5 mM Tris/HCl

6.5.3 Oocyte Removal

1. Remove the appropriate amount of ovarian tissue surgically from one side of the frog.

Please refer to standard protocols on this subject (Stühmer and Parekh 1995).

2. Transfer the ovarian lobes into a new large Petri or cell culture dish

(for example 100 mm Falcon) filled with Barth’s without Ca

6.5.4 Collagenase Digestion and Defolliculation

Ovarian tissue contains immature and mature oocytes, as well as connective tissue from which

the oocytes must be separated. Ovarian tissue should be removed completely by collagenase

digestion. Oocytes are enveloped in a follicle cell layer which can cause trouble when plating

oocytes into well plates. Remaining pieces of follicular tissue causes oocytes to stick to the walls.

Oocytes will not move into correct positions in the middle of a well by themselves. Additionally,

the follicular cell layer sometimes hampers the impalement by the injection needle causing

damage to the oocytes or clogging of the tip. Therefore, it is important that isolated oocytes

are completely free of the surrounding follicular cell layer.

The whole procedure should be completed after about 2 to 2.5 hours. Please adjust the

collagenase concentration if this is not the case.

1. Divide the tissue with a razor blade and a forceps into smaller, approximately 0.5 cm

large

pieces.

2. Transfer the clumps into 50 ml Falcon tubes containing 40 ml of 1.5 to 2.0 mg/ml collagenase in

Barth’s without Ca

. A volume of up to 7.5 ml of tissue can be put into a single tube. For more

tissue, use additional tubes. Otherwise, the collagenase digestion would take too much time.

3. Place the tubes onto the shaker and let them shake gently at 20 °C. Check the progress after

4. 90 min (and then every 15 min), and vigorously shake the tube briefly by hand to accelerate

the process.

5. Typically 120 minutes after beginning of the treatment, practically all oocytes should be

isolated and the majority of them should have already lost their follicular cell layer. If not,

continue the collagenase treatment for a maximum of 30 min.

6. Wash the oocytes extensively with Barth’s solution (minimum of 5 times with 30 ml) to remove

the collagenase completely.

7. Then fill up the tube (approx. to 45 ml) and put it back onto the shaker for 10 minutes.

8. Change the solution to Barth’s without Ca

and put it onto the shaker again for another

10 minutes. Practically all oocytes should be defolliculated now. Shake the tube vigorously

to remove the follicle cells completely, if necessary.

9. Wash the oocytes with Barth’s solution until the supernatant is clear and free of follicular

cell layer fragments.

1 2 ... 77 78 79 80 81 82 83 84 85 86 87 ... 90 91

Comments to this Manuals

No comments

Multichannel-systems Roboinject Manual User Manual Page 82

Comments to this Manuals

Related products and manuals for Equipment Multichannel-systems Roboinject Manual