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Neuronal Cell Dissociation Protocol

来源:生物谷  2009-3-11   访问量:6524
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 From  the lab of Lucien T. "Tres" Thompson, Ph.D. The University of Texas at Dallas

Edited by Barbara Gibbons (2004).                                                              

 Adapted from protocol used for immunofluorescent detection in the lab of

Dr. Santosh D’Mello, Molecular Cell Biology UTD (1999).

 

 

INTRODUCTION

         Neural tissue extractions can be studied as living organotypes such as slices, or as dissociated cells that can be cultured for recording. Fixation processed dissociated cells are commonly used for localization of proteins and other macromolecules involved in signaling and metabolism.

        During the dissociation procedure, many of the processes are lost along with the connective tissue. However, some dendrites remain intact, and limited dendritic and axonal growth can reestablish in culture. Unlike cell lines that continue to multiply, healthy neurons do not undergo further division and have limited viability. Glial cells do continue to proliferate, and can quickly overgrow and compromise the value of the neuron population. To prevent glial overgrowth, the culture or plated cells are treated with an anti-mitotic agent to prevent replication of glia. Dish cultures, maintained by regularly changing the medium, provide a unique recording preparation that continue to be viable for up to one week. For immunostaining purposes, the cells are cultured briefly in wells containing pretreated plates that the cells adhere to. The plated cells can then be easily manipulated for immunoprocessing.       

 

 

MATERIALS

 

Poly-d-Lysine treated plates (sterile round glass coverslips)

Dish with wells size appropriate for plates

BSA (Bovine Serum Albumin)

HEPES

MgSO4

Glutamine

Gentamycin

FCS  (Fetal Culture Serum)

ARA-C anti-mitotic agent

Ethanol

Trypsin

Trypsin inhibitor

DNAse

0.1M CaCl2

4% paraformaldehyde in 0.1M PBS

0.2M PBS (diluted from 10X stock)

NaCl

KCl                                                                          

125 ml flask

1 L flask

2 L flask                                                                                                          

50 ml centrifuge tubes with holder

15 ml centrifuge tubes with holder

1 ml centrifuge tube

Ridged Erlenmeyer flask

Pipette  (low µ)

Pipette for transfer of dissociation solutions (1-20 ml)

Cotton-plugged pasteur pipette

Syringe

2 large filters

Balance

Centrifuge

Orbital shaker

Hemocytometer

 

 

SOLUTIONS

 

1. KREBS buffer stock 10X

   • Measure out and add to a 2 L flask the following:

        2.237 g KCl

        1.565 g MgSO4 (anhydrous)

        1.711 g NaH2PO4•7H2O

      Add dH2O to 1 L mark and stir on stir plate.

   

  2.  Serum

       Add 10 ml KREBS to 125 ml flask and dilute with dH2O to 100 ml mark.

     • To the diluted KREBS add:

          300 mg BSA

          100 µl 1.2M MgSO4.

          1ml 1M HEPES.

       • Let stand for 10-15 min with occasional mixing.

       *** Be careful not to agitate the flask too much; the albumin has a tendency

              to stick to the glass and not dissolve.***

 

3. 0.1M PBS 

        • To 250 ml of 0.2M PBS add:

             43.8 g NaCl

             1 g KCl

          Mix well and add dH2O to a final volume of 500 ml.

 

4. Dissociation solutions

 

     Label three 50 ml centrifuge tubes: #1, #2, #3.

     Label two 15 ml centrifuge tubes: #4, #5.    

     Add to the tubes the following ingredients in this order:

 TUBE #1     TUBE #2  TUBE #3  TUBE #4 TUBE #5 
 25 ml KREBS   25 ml KREBS   21 ml KREBS  15 ml KREBS  12.5 ml KREBS
   9-10 mg Trypsin  2.1 mg DNAse  1.2 mg DNAse  12.5 µl MgSO4
     21 µg MgSO4    7.8 mg Trypsin inh.  12.5 µl CaCl2
       19 µg MgSO4  
                                                              

    Let solutions stand in capped tubes for 15 min or more with frequent

    gentle mixing.

          Then add 4ml of solution #4 to solution #3.

          Filtering:

              Under the hood, with a syringe and two large filters, remove plunger and hook

              filter onto bottom of syringe. Filter solutions in numerical order, changing filter

              after #3.

         

               ***Solutions can be frozen (-20ºC) after this step for later use.***

 

 

 

PROCEDURE

 

  1. Dissect and remove brain tissue. Separate the tissue of interest.

 

  1. Pulverize to a homogenous paste using scalpel in a swift up and down motion.

 

  1. Add the paste to tube #1, by drawing up some of the solution in a cotton-plugged pasteur pipette, and with rapid ejections, wash the tissue into the #1 tube.

 

  1. Spin tube #1 (4000 rpm) for one minute. Discard supernatant by pipetting and suctioning.

 

  1. Add 25 ml of solution #2 to tube #1. Transfer to ridged Erlenmeyer flask. Shake in orbital shaker for 10 min at 150 rpm, and transfer to a fresh 50 ml tube.

 

      6.   Meanwhile, make the culture medium (≈ 180 ml final volume):

               • Add 1 ml gentamycin and 5 ml glutamine to pre-made serum, and warm.

               • Warm 3.6 ml KCl.

               • Then add 20 ml FCS (≈10%) and the warm 3.6 ml KCl (18 µl per ml) to the

                   serum mix, and set aside.

 

  1. Add 25 ml of solution #3 to tube. Mix and spin immediately at 4000 rpm for 1 min.

      ***You want to see white cells in medium, not a tornado. If tornado, titrate 6 ml

             of solution #4 into tube with pipette, and spin again for 2 to 3 min.***

     

      8.    Gently remove top layer (supernatant) without disturbing pellet. Add 6 ml

             solution #4 to tube (with pellet), and transfer to fresh 15 ml tube. Suction and

             release gently 25 times with cotton-plugged pasteur pipette to separate cells.

             Then place top on tube, and allow to stand for 5 min.

           

              *** The top (80%, 80%,95%) of mixture removed in the next steps now

              contain the cells separated from connective tissue and other tissue debris.***

 

      9.    Carefully transfer upper 80% (≈ 4-5 ml) to fresh 50ml tube. Retain 15 ml tube 

             containing the remaining 20%.

 

10.     Add 4 ml of solution #4 to 15 ml tube with remaining 20%. Repeat

        suction – release separation 25 times. Leave 10 min with cap on.

    

11.     Transfer the upper 80% of this tube’s contents to the 50 ml tube holding the first

        80% transferred in step #8. Total volume should be about 8 ml. Add 10 ml of

        solution #5 and mix. Let stand for 5 min.

 

12.   Then transfer the top 95% to a 50 ml tube. There should not be a pellet at this

         point. Spin at 4000 rpm for 10 min, and discard supernatant. Remaining in the

         tube are suspended dissociated cells.

 

13.   Heat culture medium (from step 6) to 37ºC.

 

     14.   Add the content of the tube (suspended dissociated cells) to 20 ml of culture

         medium. Pipette suction 10 – 15 times to mix and separate clumps. Remember

         no bubbles. This is the cell-mix.

                                                                                                                                                                                     

 15.   Aliquot 450 µl of culture medium into 1 ml tube. Pipette 50 µl of cell-mix into

 tube of culture medium, and mix well. This is a counting sample of the final cell

         suspension.

 

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