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| − | == Scope == | + | == Usage == |
| − | 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 | + | 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. |
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| − | == Training ==
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| | 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. | | 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. |
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| − | == Carrying out work in the room ==
| + | The room must be booked in [https://bookitlab.uio.no/ BookitLab]. UiO has a [https://www.uio.no/english/services/it/adm-services/bookitlab/help/index.html BookitLab user guide]. |
| − | * The room must be booked in the [https://www.mn.uio.no/fysikk/forskning/prosjekter/laglivlab/arrangementer/calendar-cell-lab-fv431.html calendar]
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| − | * ...
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| − | == List of things to check regularly in 431 ==
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| − | * '''<u>Sterile water:</u>''' Always at least one full bottle of autoclaved water
| + | = List of maintenance procedures = |
| − | * '''<u>75% alcohol spray:</u>''' Always at least one full spray flask of 75% alcohol
| + | === Preparation of solutions === |
| − | * '''<u>DMEM ++:</u>''' Always one half full bottle of DMEM supplied with extras | + | * [[Dulbeccos Modified Eagle's Media, DMEM adding extras | DMEM]] |
| − | * '''<u>Yellow waste container:</u>''' full, clean then thoroughly before taking them in the lab
| + | * [[Diluted PBS | PBS]] |
| − | * '''<u>sticky mat:</u>''' Check that sticky mat sticks
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| − | * '''<u>Vacusafe:</u>''' Empty when it is more than half full | |
| − | * '''<u>Water in incubator:</u>''' Once a week, refill sterile water in incubator. Need: 1-2 l.
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| − | * '''<u>Diluted PBS:</u>''' When the diluted PBS bottle is less than half full prepare another
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| − | * '''<u>List:</u>''' Check the list of things to buy (on the wall in the fore-room)
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| − | * '''<u>CO2:</u>''' Check the CO2 level in bottles. Reorder bottle if necessary.
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| − | * '''<u>Pasteur pipettes:</u>''' Always at least 20 sterile Pasteur pipettes in hood, if not: autoclave new
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| − | * '''<u>Coats:</u>''' Every 3 weeks: wash coats
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| − | * '''<u>Water:</u>''' Change water in distilled water small containers
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| − | == Cleaning == | + | === Lab maintenance === |
| − | === Weekly cleaning === | |
| − | Every week the lab has to be cleaned.
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| − | * Move all loose pieces of equipment from lab benches into cupboards and drawers
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| − | * Throw away all unmarked consumables
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| − | * Wipe all surfaces (benches, PC, microscope, ...) with antibac
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| − | * Clean '''LAF bench/hood''' with antibac
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| − | ** Leave only minimum equipment in hood
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| − | ** Remove the base of the bench and clean in the sink
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| − | ** Clean under the base
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| − | ** Clean the inside of the glass sides (windows)
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| − | ** Reassemble
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| − | ** Turn on the UV light
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| − | * Clean '''Incubators''' (if needed)
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| − | ** Clean one incubator at the time
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| − | ** Move all cell flasks etc to the other incubator
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| − | ** Remove all loose parts and clean all parts and surfaces with antibac
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| − | ** When needed, decontaminate the incubators (with heat or with peroxide, follow instructions in the manuals).
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| − | * Clean '''fridge'''
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| − | ** Remove all unmarked or overdue (check date) containers to trash
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| − | * Clean inside the centrifuge
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| − | # First clean with distilled water
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| − | # Then clean with ethanol
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| − | # Clean all tube holders with ethanol
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| − | * Mop the floor
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| − | * Spray "what is touched" (handles etc...)
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| | | | |
| − | == Vacusafe ==
| + | * [[Autoclaving]] |
| − | * Step 1: Add 50 ml chlorine (around this amount) to the bottle. | + | * [[Vacusafe]] |
| − | * Step 2: find one autoclave bag in v420, pour all into the bag and tight the bag. | + | * [[Incubator]] |
| − | * Step3: put the waste bag into yellow container. | + | * [[Cleaning V431 | Cleaning]] |
| − | | + | * [[Waste disposal]] |
| − | == [[Maintenance|Other maintenance procedures]] ==
| + | * [[Standard items | List of standard items]] |
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| | = Cell culturing = | | = Cell culturing = |
| − | == Hela cells == | + | === Cell lines === |
| − | | + | * [[Culturing Hela cells|Hela]] |
| − | === Passaging === | + | * [[Culturing MDCK cells|MDCK]] |
| − | | + | * [[Culturing RPE cells|RPE]] |
| − | | + | * [[BV-2]] |
| − | * 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. | + | * [[Muscle cells]] |
| − | * 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.
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| − | * 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.
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| − | * If you are keeping the same flask:
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| − | ** 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.
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| − | * If you are moving to a new flask:
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| − | ** 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. | |
| − | | |
| − | == Muscle cells ==
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| − | In the Bioactuator project, we use C2C12 myoblasts. For more information about C2C12 myoblasts click on the following link: https://en.wikipedia.org/wiki/C2C12#:~:text=C2C12%20is%20an%20immortalized%20mouse%20myoblast%20cell%20line.&text=C2C12%20cells%20are%20used%20to,to%20explore%20mechanistic%20biochemical%20pathways.
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| − | | |
| − | We use the following cell culture protocol for the C2C12 cells:
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| − | | |
| − | '''C2C12 cells protocol'''
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| − | | |
| − | '''I-General information'''
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| − | | |
| − | 1. The base medium for this cell line Dulbecco's Modified Eagle's Medium (DMEM, Sigma D6429) with 10% fetal bovine serum (FBS) and 1% Penicillin/Streptomycin.
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| − | | |
| − | 2. The seeding density should be 5.0 X10<sup>3</sup>viable cells/cm<sup>2</sup>
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| − | | |
| − | 3. IMPORTANT - DO NOT ALLOW CULTURES TO BECOME CONFLUENT.
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| − | Cultures must not be allowed to become confluent as this will deplete the myoblastic population in the culture.
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| − | | |
| − | Myotube formation is enhanced when the medium is supplemented with 10% horse serum instead of fetal bovine serum.
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| − | | |
| − | '''II-Trypsinizing or passaging the cells'''
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| − | | |
| − | 1. Remove and discard culture medium. Rinse the cells with PBS twice.
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| − | | |
| − | 2. 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.
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| − | | |
| − | 3. Add 8.0 mL of complete growth medium and dispense cells by gently pipetting.
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| − | | |
| − | 4. Tranfer the cell suspension into 5a 0 ml tube.
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| − | | |
| − | 5. Centrifuge the cell suspension to make cell pellet (300g, 3 min). Remove the supernatant and re-suspend the cells in 10 ml of culture medium.
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| − | 6. Add appropriate aliquots of the cell suspension to new culture vessels.Inoculate at a cell concentration between 1.5 X 10<sup>5</sup> and 1.0 X 10<sup>6</sup> viable cells/75 cm<sup>2</sup> bottle.(Corning® T-75 flasks (catalog #430641) are recommended for subculturing this product.
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| − | | |
| − | 7. Incubate cultures at 37°C.
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| − | | |
| − | 8. Medium Renewal: Every two to three days (see the medium colour)
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| − | == Collagen for 3D or coating surfaces == | + | === Other === |
| − | 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.
| + | * [[Collagen | Collagen for 3D or coating surfaces]] |
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| − | [https://ibidi.com/cell-culture-microscopy/107-collagen-type-i-rat-tail.html#/110-concentration_volume-5_mg_ml_4_x_5_ml Type 1 Collagen from Ibidi], '''[https://ibidi.com/img/cms/products/cells_reagents/R_5020X_CollagenI/IN_5020X_CollagenI_05mg.pdf 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). | + | === Protocols === |
| | + | * [[Micropatterning on hydrogels]] |
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.
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.
