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dissociated neurons for patch clamp or Immunohistochemistry

来源:生物谷  2009-3-11   访问量:5435
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From  the lab of Lucien T. "Tres" Thompson, Ph.D. The University of Texas at Dallas

 

Protocol for Dissociated Neurons

 

Purpose:  This protocol uses trypsin, a digestive enzyme, to degrade proteins in brain tissue.  This will produce a one cell thick layer of dissociated cells for further studies in patch clamp recording or immunohistochemistry.

 

Materials:

            Hippocampal brain slices from rat

            Lypholized trypsin enzyme

            Buffer solution, oxygenated and chilled (Usually a TRIS or HEAPS buffer.)

            Bovine Serum Albumin (BSA)

            Glass Pasteur pipettes with rubber bulbs

            Bunsen burner

            50 mL centrifuge tubes (should be sterile)

35-mm plastic Petri dish with lid, preferably coated

           

1)      Prepare hippocampal brain slices from rat.  To obtain specific isolated cell types, dissect out the hippocampal portion of interest.  (CA1, CA3, DG, etc…)

2)      Prepare buffer.  aCSF.  Oxygenate and chill.

3)      Prepare 50 mL of trypsin enzyme in a centrifuge tube.

a.       For a low concentration, mix 0.5 mg trypsin in 50 mL buffer solution.  (10 μg / mL)

b.      For a higher concentration, mix 1.0 mg trypsin in 50 mL buffer solution (20 μg / mL)

 

Safety Note:  Avoid getting trypsin on your skin, as trypsin will digest any protein it comes into contact with.

4)      Gently swirl the brain in trypsin for about 15 minutes.

5)      Cells will start to clump in the trypsin, indicating digestion.

6)      After 15 minutes, dump BSA into the trypsin solution to saturate the enzyme and stop the digestion

7)      Break the narrow tip off the end of a Pasteur pipette.  Fire-polish the broken end over a Bunsen burner flame to smooth the edges.  Be careful that the opening does not close.  The flame temperature may need to be adjusted to get consistently good results.  Turn off Bunsen burner when done.

Safety Note:  The tip will be very hot when removed from the flame.  Allow to cool before touching.

8)      Repeat step 5 for several pipettes.  The final pipette tips should have several different-sized openings.

9)      Tritaration Step:

a.       Arrange pipettes by diameter size of their tip openings

 

b.      Starting with largest pipette, suction and release tissue to break it up.

 

                                                              i.      Drawn tissue all the way into the upper portion of the pipette.

                                                            ii.      Allow cells to settle to the bottom of the tube between draws.

                                                          iii.      Concentrate on drawing the largest chunks of tissue from the bottom of the tube.

                                                          iv.      Repeat until no further tissue disintegration is seen.

 

c.       Repeat step 9) b. with progressively smaller pipettes until all pipettes have been used.

 

d.      The final liquid will look cloudy.

 

10)  Plate the cells onto a Petri dish, pouring just enough to spread into a thin layer.  Coated plates will disperse the cells more easily.

 

11)  Cover the Petri dish with a lid and let it sit for one hour.  The cells will anchor to the dish.  Keep the Petri dish chilled during this period.

 

12)  Examine the dish under a microscope or IRDC scope for individual cells.

 

Discard pipettes in sharps or glass disposal.  Discard centrifuge tubes with regular waste.

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