Difference between revisions of "Cell lab 431"
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=== Passaging === | === Passaging === | ||
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* Remove and discard culture medium using the vacusafe and pasteur pipette. Rinse the cells with PBS (around 10mL) and remove it. This is because the Trypsin is inactivated by FBS in the media. | * Remove and discard culture medium using the vacusafe and pasteur pipette. Rinse the cells with PBS (around 10mL) and remove it. This is because the Trypsin is inactivated by FBS in the media. | ||
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* Put your name (if not already present) the date and increase the passage number on the flask and put them back in the incubator. | * Put your name (if not already present) the date and increase the passage number on the flask and put them back in the incubator. | ||
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== Collagen for 3D or coating surfaces == | == Collagen for 3D or coating surfaces == | ||
Revision as of 13:17, 3 April 2022
Contents
Scope
The purpose of this SOP is to describe routines for the use of cell culture lab, room 431 on the 4th floor of west wing in the Physics building
Training
All users of the cell lab must have direct training from the room responsible person before room is used. New user must sign the completed training form.
Carrying out work in the room
- The room must be booked in the calendar
- ...
List of things to check regularly in 431
- Sterile water: Always at least one full bottle of autoclaved water
- 75% alcohol spray: Always at least one full spray flask of 75% alcohol
- DMEM ++: Always one half full bottle of DMEM supplied with extras
- Yellow waste container: full, clean then thoroughly before taking them in the lab
- sticky mat: Check that sticky mat sticks
- Vacusafe: Empty when it is more than half full
- Water in incubator: Once a week, refill sterile water in incubator. Need: 1-2 l.
- Diluted PBS: When the diluted PBS bottle is less than half full prepare another
- List: Check the list of things to buy (on the wall in the fore-room)
- CO2: Check the CO2 level in bottles. Reorder bottle if necessary.
- Pasteur pipettes: Always at least 20 sterile Pasteur pipettes in hood, if not: autoclave new
- Coats: Every 3 weeks: wash coats
- Water: Change water in distilled water small containers
Cleaning
Weekly cleaning
Every week the lab has to be cleaned.
- Move all loose pieces of equipment from lab benches into cupboards and drawers
- Throw away all unmarked consumables
- Wipe all surfaces (benches, PC, microscope, ...) with antibac
- Clean LAF bench/hood with antibac
- Leave only minimum equipment in hood
- Remove the base of the bench and clean in the sink
- Clean under the base
- Clean the inside of the glass sides (windows)
- Reassemble
- Turn on the UV light
- Clean Incubators (if needed)
- Clean one incubator at the time
- Move all cell flasks etc to the other incubator
- Remove all loose parts and clean all parts and surfaces with antibac
- When needed, decontaminate the incubators (with heat or with peroxide, follow instructions in the manuals).
- Clean fridge
- Remove all unmarked or overdue (check date) containers to trash
- Clean inside the centrifuge
- First clean with distilled water
- Then clean with ethanol
- Clean all tube holders with ethanol
- Mop the floor
- Spray "what is touched" (handles etc...)
Vacusafe
- Step 1: Add 50 ml chlorine (around this amount) to the bottle.
- Step 2: find one autoclave bag in v420, pour all into the bag and tight the bag.
- Step3: put the waste bag into yellow container.
Other maintenance procedures
Cell culturing
Hela cells
Passaging
- Remove and discard culture medium using the vacusafe and pasteur pipette. Rinse the cells with PBS (around 10mL) and remove it. This is because the Trypsin is inactivated by FBS in the media.
- Add 2.0 mL of Trypsin-EDTA solution to flask and put it back in the incubator and wait about 5 minutes. Tap the flask and observe cells under an inverted microscope until cell layer is dispersed.
- Add 8.0 mL of complete growth medium, this will stop the action of trypsin. Pipette up and down (gently) a few times to break the clusters of cells.
- If you are keeping the same flask:
- Pipette away and discard a certain amount of media. The exact amount depends on the desired density of the passaged cells.
- Add fresh media to the flask.
- If you are moving to a new flask:
- Pipette the amount of cells you want in a new flask (less than the total if you want to decrease the density of cells in your flask).
- Add some media to the new flask (12 - 15 mL in T75 or 5 mL in T25 - total).
- Put your name (if not already present) the date and increase the passage number on the flask and put them back in the incubator.
Collagen for 3D or coating surfaces
Type I Collagen is a major structural component of the extracellular matrix (ECM). Therefore, this fibrous protein is often used in three-dimensional (3D) collagen gels that simulate the in vivo cell environment better than the traditional 2D systems. Additionally, Collagen I is ideal for coating surfaces, as it can form thin layers for culturing cells.
Type 1 Collagen from Ibidi, Protocols for use. (bought through Inter Instrument https://interinst.no/. The product number is IB 50202 (4x5ml 5 mg/ml), price 3226,- + MVA Feb 2021).
