# Goal/Experiment:
To culture adrenal chromaffin cells derived from Sprague Dawley rats aged 7 to 12 days.
# Adrenal Chromaffin Cell Cultures
**Authors:**
Ellen Kantar<sup>1</sup>, David Sulzer<sup>1</sup>
<sup>1</sup>Columbia University
**Date:**
May 18, 2021
**DOI:**
[dx.doi.org/10.17504/protocols.io.bpkzmkx6](https://dx.doi.org/10.17504/protocols.io.bpkzmkx6)
## Abstract
This protocol details the culturing of adrenal chromaffin cells from rats. Adrenal glands from 7- to 12-day-old Sprague Dawley rats are dissected in ice-cold Hanks Balanced Salt Solution (HBSS).
### Keywords
rat-derived cultures, cell culture, adrenal, chromaffin, rat
### License
This protocol is distributed under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/), permitting unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
## Guidelines
### Suggestions for Plating Density for Rat and Mouse CC Cultures:
- 3 × 10-day-old rat pups – 8 dishes
- 5 × 10-day-old rat pups – 16 dishes
- 2 adult mice – 12 dishes
- 10 × 10-day-old mouse pups – 12 dishes
## Materials
### Reagents:
- **Ketamine (Anaesthetic) if decapitation is not an approved protocol: Ketase®**
*Vendor*: FORT DODGE
*Catalog #: NDC-0856-2013-01*
- **L-Glutamine solution, 200 mM**
*Vendor*: Sigma
*Catalog #: G2150*
- **Penicillin-Streptomycin**
*Vendor*: Sigma
*Catalog #: P0781*
- **Fetal Bovine Serum, qualified, heat-inactivated, United States**
*Vendor*: Thermo Fisher
*Catalog #: 16140063*
- **DMEM - Low Glucose**
*Vendor*: Sigma
*Catalog #: D5546*
- **HBSS, no calcium, no magnesium, no phenol red**
*Vendor*: Thermo Fisher
*Catalog #: 14175095*
- **Collagenase Type I**
*Vendor*: Worthington Biochemical Corporation
*Catalog #: LS004197*
- **Deoxyribonuclease I**
*Vendor*: Worthington Biochemical Corporation
*Catalog #: LS002006*
### Preparation:
#### CC Media (200 mL):
- 2 mL L-Glutamine
- 240 µL Pen-Strep
- 20 mL Fetal Bovine Serum, heat-inactivated
- 180 mL DMEM
#### Trituration Solution (110 mL):
- 10 mL HBSS
- 30 µL DNase stock (final concentration 0.02%)
- 100 µL Fetal Bovine Serum, heat-inactivated
#### Preparation of DNase I Stock Solution:
- Reconstitute with HBSS to a concentration of 2000 U/mL (e.g., a vial with 20 mg and 3364 U/mg is reconstituted with 33.64 mL HBSS).
- Store in 500 µL aliquots at -80 °C.
### Safety Warnings:
For hazard information and safety warnings, please refer to the SDS (Safety Data Sheet).
## Protocol Steps:
1. **Animal Preparation:**
- Animals are decapitated (anaesthetize the animals with Ketase® KETAMINE, FORT DODGE® NDC-0856-2013-01 if decapitation is not an approved protocol).
2. **Body disinfection and pinning:**
- Decapitate and pin the body belly-down. Spray with 70% ethanol.
3. **Back skin opening:**
- Cut the skin along the spinal column (easier starting from the neck) and pull it out to both sides, using scissors to separate the skin from underlying tissue. The back of the body is now open.
4. **Removal of adrenal glands:**
- Grab the vertical column with forceps and pull it up while cutting along both sides through the ribs. Alternate cuts on each side as you work back. Once the diaphragm is reached, open the scissors and place onto the diaphragm approximately 1/3 of the way up from the bottom. Pull the spine up while holding the diaphragm down. Expose the abdominal cavity showing two kidneys with adrenal glands on top.
5. **Isolation of adrenal glands:**
- Remove the glands with fine forceps (preferably curved), pinch off the tissue under the glands, and pull up. Place into ice-cold HBSS (Ca²⁺/Mg²⁺-free).
6. **Removal of capsule:**
- Adrenal glands are encased by adipose tissue and a capsule. Remove using two fine forceps, pull the capsule open like a bag, and use the other to roll off the gland. Cut the adrenal glands in half or thirds depending on their size.
7. **Cleaning and digestion:**
- After several washes with HBSS (using a sterile plastic transfer pipette), digest the tissue with Collagenase I in 10 mL HBSS, Ca²⁺/Mg²⁺-free, (250-350 U/mL, Worthington) for about 30:00 at 37 °C with stirring. Stop the digestion once the solution becomes cloudy.
8. **Rinse and triturate:**
- Rinse the digested tissue with HBSS and triturate gently in a solution containing 1% heat-inactivated fetal bovine serum and 0.02% DNase I. Use large bore tech-tips for trituration, medium bore if needed.
9. **Centrifuge and resuspend cells:**
- Centrifuge dissociated cells at 1000 x g, 00:03:00 to form a pellet. Resuspend in culture medium with DMEM, 10% fetal bovine serum, 50 U/mL penicillin, 50 µg/mL streptomycin, and 2 mM Glutamine.
10. **Plating cells:**
- Plate cell suspension on poly-D-lysine and laminin-coated glass wells in 50 mm dishes (cells from 5 rat 10-day-old pups onto 16 dishes), and after 2:00:00, flood dishes with culture medium (3 mL per dish).
11. **Incubation:**
- Maintain cells in a 5% CO₂ incubator at 37 °C. Conduct all measurements between 1 and 7 post-plating days.
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Please refer to the "Ventral Midbrain Cultures" protocol for instructions on how to prepare and coat dishes.
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