CNNS experiment protocol
From Neural Wiki
Protocol for setting up cultured networks at CNNS
Contents |
[edit] Preparation
1. Check that the station you wish to use is properly equipped. There should be a working heater, aalborg air supply, amplifiers, computer, and water pump
2. Make sure that there are autoclaved chambers available. Check that these chambers match the type of Microelectrode array you will be using. For example, if you are using a plate that has one culture on a single array (MEP 3 or 4), make sure the chamber has a singe opening. If the plate has two cultures on two arrays (MEP 5), then check that there are chambers with two openings.
3. Start warming up the microscope by turning on the stage heaters (if so equipped)
4. Place the following items underneath the laminar flow hood:
- chamber
- heater plate (black or blue plate with resistors on it)
- screws that hold the chamber to the heater plate
- pair of tweezers
- 3 ml syringe or disposable suction pipette
- IF you will be doing a medium change, place the new medium under the hood as well
[edit] Culture Selection
1. Before entering the clean room put on gloves, sleeves, mask, hair net, shoes, and then spray down with 70% ethanol
2. Look on the back wall where information about what is in each incubator is hung. Figure out which incubator you would like to look for cultures in. You want to minimize your time in the clean room, so decide what you are looking for and where it is before you enter the clean room.
3. After entering the clean room, open up the incubator you wish to select a culture from and find 2-3 cultures for examination under the microscope. BE CAREFUL, DON'T CONTAMINATE CULTURES. DO NOT OPEN THE LIDS!
4. Take the cultures you chose over to the clean room microscope, place one of the cultures on the stage, and turn on the light.
5. Look for good cell adhesion, nice looking processes, cell bodies and most importantly CONTAMINATION!
- If the culture is contaminated, set it aside for the moment, but when you leave the clean room place the culture in one of the sinks, and pour bleach on it
6. Look at all of the cultures you brought out and once you find a culture that you like, return the other non contaminated cultures to the incubator (spray them with alcohol first)
- If none of the cultures look good, then set all of them aside for bleaching (no need to keep feeding them), and go back to step 3.
7. Turn off the microscope, make sure to turn down the light first before turning it off (this helps save the bulb).
8. Leave the clean room, set the culture you will be doing an experiment on in the little window between rooms, and remove your protective gear.
[edit] Assembly
- Assembly should be done in a prompt manner, as the PH and temperature are in a constant state of change without life support
1. Remove the culture from the window and place it under the laminar flow hood
2. Arrange everything under the hood such that you will not have to reach over the culture or the chamber once you open up their respective petri dishes
- Picture 1 shows the components that you will need. It is advisable that you do not remove the chamber from its petri dish before it is needed as shown in the picture. Such actions could lead to contamination
3. Remove the lid from the culture's petri dish
4. Suck out the majority of the medium with the syringe or pipette. BE careful to suck from the outside, and not over the neurons as it is possible to suck up your culture!
5. Remove the MEP from the bottom petri dish using the tweezers (it is held in place with silicone grease)
6. Place the MEP onto the heating plate
- This is illustrated in Picture 2. If you look carefully you will note that the medium had not yet been sucked out. As long as the medium is removed before the next step, it is acceptable
7. Remove the red silicone gasket from the MEP
8. Place the chamber over the MEP and screw it into place. MAKE SURE THAT the chamber is laying flat, so that the o-ring seals. Do not tighten one side more than the other, this will lead to leaks
9. Replace the medium that you took out, or put in fresh medium
10. Take a 100 mL sample for PH and Osmolarity analysis
11. Put the chamber cap on
- Picture 3 shows a fully assembled chamber that is ready to be placed on the microscope stage
12. Carry it to the microscope stage and set it down there
14. Plug the CO2, and air mixture into the cap (coming from the aalborg). Make sure that it is flowing. No CO2/air mixture=dead culture
15. Plug in the heater plugs and the thermocouple
16. Turn on the heater
17. Use filter paper to clean up any excess medium by running it under the lip of the MEP until it does not pick up any more medium. IF medium keeps coming out, you probably have a leak
- If you think that you have a leak, try tightening down the chamber, but first make sure that it looks level. Most leaks occur when one side has been tightened down more than the other. Other possibilities are bad O rings, or a cracked MEP.
18. Find two zebra strips, which should be living in a vial of ethanol
19. Wipe them down with a Q-tip. Do not touch them with bare hands, they need to remain clean to conduct signals
20. Clean the MEP fingers with a Q-tip. These are the glass part of the MEP that are protruding from beneath the chamber
21. lay the zebra strips on the fingers
22. Move the amplifiers over the zebra strips, and screw them down using the pressure bars
- This should look something like Picture 4
[edit] Recording
1. Turn on the Harvey box
2. If the computer is not turned on, go ahead and turn it on
3. Turn on the preamplifiers. The switch is located on the Harvey box
4. Turn on the speaker array
5. Go to the Plexon folder on the desktop of the computer
6. Double click on the server, and wait until it says program loaded
7. Double click on the Plac
8. Double click on the Sort Client
9. Find the Start button in Sort Client and click it
10. Go to global settings and raise the top number to 1000 and the second number to 300
11. On the Plac, raise gain to 10000 and then click on the global option
12. Sort your units
Now you have completed all of the steps necessary to begin recording an experiment
[edit] Experiment types
Native activity (for analysis)
Pharmacology (studying network states after pharmacological additions)
