Abbreviations: ACCs - Adrenal Chromaffin Cells, E.Soln - Enzyme Solution, E.DMEM - Enriched Dulbecco's Modified Eagle Medium
<ol>
<li>Preparation<ol>
<li>Add papain to desired quantity of E. Soln (40 units papain/ 1ml E. Soln.).</li>
<li>Activate papain with carbogen for about 15 minutes on ice.&nbsp;Oxygenate Locke's buffer on ice as well. </li>
</ol></li>
<li>Dissection<ol>
<li>Use 8-10 week mouse.</li>
<li>Prior to digestion remove oxygenated Locke's buffer and place on ice.</li>
<li>Refer to video for dissection procedure.</li>
<li>Once excised, place glands in oxygenated Locke's buffer. </li>
</ol></li>
<li>Preparation of Gland<ol>
<li>Remove fat from gland.</li>
<li>Strip cortex from gland. </li>
</ol></li>
<li>Enzymatic Digestion<ol>
<li>Sterile filter oxygenated papain.</li>
<li>Make four pools of oxygenated papain on a petri dish.<ol>
<li>3 pools should be about 100uls.</li>
<li>1 of the pools should be about 400uls.</li>
</ol></li>
<li>Wash medullae through pools<ol>
<li>1st through the 3 100ul pools.</li>
<li>Then through the 400ul pool</li>
<li>Pippette 300ul E.Soln and medulla pieces from 400ul pool into microfuge tube.</li>
<li>Qrap parafilm on microfuge tube and place in 37&deg;C water bath for 20min.</li>
<li>Remember to continue to oxygenate remaining E.Soln containing papain.</li>
<li>After 20 minutes sterile filter more E.Soln.</li>
<li>Replace old E.Soln bathing medullae with newly filtered E.Soln. </li>
</ol></li>
</ol></li>
<li>Triturations<ol>
<li>Aspirate and discard E.Soln.</li>
<li>Wash medullae in 1ml E.DMEM.</li>
<li>Aspirate and discard E.DMEM.</li>
<li>Add 300ul E.DMEM and triturate 20X with 1ml tip.</li>
<li>Cut 200ul tip with sterile razor blade.</li>
<li>Triturate 5-10X with cut 200ul tip.</li>
<li>Discard E.DMEM bathing medullae pieces.&nbsp; Medium will contain debris.</li>
<li>Add 300uls fresh E.DMEM to medullae pieces.</li>
<li>Triturate with 200ul tip 20x</li>
<li>Pippette E.DMEM to microfuge tube withough medullae pieces.&nbsp; Medium will contain free ACCs.</li>
<li>Add 300uls fresh E.DMEM to medullae pieces.</li>
<li>Triturate medullae with a 23.5 gauge syringe needle 20x.</li>
<li>Combine E.DMEM from both triturations.&nbsp; Medullae pieces should not be present and should now have a featherlike appearance. </li>
</ol></li>
<li>Plating<ol>
<li>Microfuge E.DMEM from the last two trituration steps for 2.5 minutes at 3.7g s.</li>
<li>Resuspend pellet in 50uls-200 E.DMEM, depending on subsequent experiments.</li>
<li>Plate 10-30uls on a glass coverslip.</li>
<li>Wait 15 minutes for cells to adhere.</li>
<li>Add 750uls of E.DMEM. &nbsp;</li>
</ol></li>
</ol>

The authors have nothing to disclose.

mouse adrenal chromaffin cell isolation

Adrenal medullary chromaffin cell culture systems are extremely useful for the study of excitation-secretion coupling in an in vitro setting.  This protocol illustrates the method used to dissect the adrenals and then isolate the medullary region by stripping away the adrenal cortex.  The digestion of the medulla into single chromaffin cells is then demonstrated.

Watch this Scientific Journal Video about Mouse Adrenal Chromaffin Cell Isolation at JoVE.com

Mouse Adrenal Chromaffin Cell Isolation

Adrenal medullary chromaffin cell culture systems are extremely useful for the study of excitation-secretion coupling in an in vitro setting. This protocol illustrates the method used to dissect the adrenals and then isolate the medullary region by stripping away the adrenal cortex. The digestion of the medulla into single chromaffin cells is then demonstrated.

Adrenal medullary chromaffin cell culture systems are extremely useful for the study of excitation-secretion coupling in an in vitro setting.  This ...

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17.0K Views.  University of California, Irvine (UCI). Note: This video does n

Video: Mouse Adrenal Chromaffin Cell Isolation

Mouse Dorsal Forebrain Explant Isolation

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Isolation and Culture of Post-Natal Mouse Cerebellar Granule Neuron Progenitor Cells and Neurons 

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Isolation and Culture of Post-Natal Mouse Cerebellar Granule Neuron Progenitor Cells and Neurons

Here we present a method to isolate and culture cerebellar granule neuron progenitor cells and cerebellar granule neurons from postnatal mouse.

The cerebellar cortex is a well described structure that provides unique opportunities for studying neuronal properties and development1,2. Of the ...

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Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

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Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of ...

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Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...