Protocols_CellCulture_Subculturing Protocol_V1 - BackmanLab/BackmanLab.github.io GitHub Wiki

Procedure: Subculturing Adherent Cells

SOP Prepared by: Srutartha Bose Version: 1.0

1. Scope

The purpose of this SOP is to describe how to passage adherent cells.

2. Description

This protocol aims to describe the procedure for passaging adherent cells by using respective cell culture reagents and maintaining strict conditions required for optimal cell growth in a BSL2 tissue culture environment.

3. Personal Protective Equipment

• Gloves

• Lab Coat

4. Potential Hazards + Safety Precautions

• Nitrile gloves must be used at all times.

• Avoid bringing hands close to the face or to any exposed body part.

• All cell culture suspensions and reagents must be opened only in the sterile tissue culture hood.

• Any spillage must be cleaned immediately with either 70% Ethanol or 10% Bleach.

• Cells to be discarded must be immersed in at least 10% Bleach prior to disposal.

5. Materials and Reagents

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1. Equipment

• Centrifuge (Allegra X12 - shared bench in B715)

• VWR Water Bath (by the sink in Tissue Culture Room)

• Sterile Hood (LABCONCO Class II Biosafety Cabinet - Tissue Culture Room)

• Primary Cell Incubator (VWR - smaller incubator on the left of Biosafety Cabinet)

• Secondary Incubator (SANYO CO2 Incubator - beneath the VWR Incubator)

• Microscope (LABOMED TCM400 Microscope - cell counting desk in Tissue Culture room)

• Cell Counter (Invitrogen, Countess II -- cell counting desk in Tissue culture Room)

• Refrigerators (4$℃$, 0$℃$)

= Subculturing Adherent Cells (Cont.) =

2. Materials

• Sterile pipettes and tips (located in Biosafety cabinet)

• Disposal bottle (in the hood)

• Biohazard disposal bag (in the hood)

• Gun pipette

• 15 ml and 50 ml centrifuge tubes

• 0.5 ml centrifuge tubes

• Flasks with ventilated caps

• Kim wipes

• Countess Slides

3. Reagents and Solutions

• Cell Culture Media (supplemented with 10% FBS + 1x Pen/Strep)

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• For 500 ml of media, remove 50 ml of media from the bottle and substitute it with 50 ml FBS and then add 5 ml of Pen/Strep)

NOTE 1: Keep FBS (aliquots of 50 ml) and Pen/Strep (aliquots of 10 ml) refrigerated at 0$℃$ when not in use. Aliquoting is done to avoid repetitive thawing which may degrade the reagents' components.

• 0.25% Trypsin EDTA (1x)

• PBS pH 7.4 (GIBCO)

• 10% Bleach solution

NOTE 2: Wear lab coat to avoid bleach from ruining clothes

= Subculturing Adherent Cells (Cont.) =

6. Procedure

Preparation

NOTE 3: For passaging your own cell line, it is recommended that the product's protocol is followed as given as different cell lines may have different requirements.

1. Before starting the procedure, ensure that all reagents are at 37$℃$ by keeping them in the hot water bath for 20 to 30 mins.

2. Make sure the inside of the hood is entirely sprayed and wiped with 70% Ethanol. Any item that goes inside the hood must be sprayed and wiped with 70% Ethanol with no exception.

NOTE 4: Reagents must not be heated beyond 37$℃$ and over 30 minutes as these will degrade the components that are necessary for optimal cell growth.

3. Check the confluency of the cells under the Microscope. Split only if they are around 70% - 80% confluent.

Washing

1. Discard the old media into the disposal bottle in the hood.

2. Perform a wash with 1 ml PBS and discard it into the disposal bottle.

Dissociation/Trypsinization

1. Add 1 ml of Trypsin (approximately 0.5 mL per 10 cm^2^) into the side of the flask. Gently rock the flask to ensure the entire surface is covered with it.

2. Place the flask in the primary incubator for approximately 1 to 2 minutes to ensure quick trypsinization.

3. Check if all cells have dissociated well by viewing the flask under the microscope. If some are still attached, gently tap the flask onto the desk a couple of times and then slightly tap it with fingers at the sides of the flask giving it a little bit of a shake.

Trypsin inactivation and removal

1. In the hood, add 3 ml of respective media (Trypsin to Media ratio must be 1:3) to the flask and wash the surface of the flask well by pipetting the suspension against it twice. Transfer this 4 ml of cell suspension into a 15 ml centrifuge tube and label it respectively.

2. Centrifuge the cell suspension at 200 g for 5 minutes in Allegra X-12 Centrifuge.

NOTE 5: Check if the centrifuge is clean and empty prior to keeping the cell tube in it. Do not forget to keep the balance tube of 4 ml of water in the slot opposite to that of the cell tube.

3. While the cells are getting centrifuged, place Trypan Blue stain, Countess slide and 0.5 ml centrifuge tube inside the hood after spraying them with 70% Ethanol

4. After centrifuge is completed, discard the supernatant and resuspend the cells in 2 ml of media.

= Subculturing Adherent Cells (Cont.) =

Cell counting

1. Take 15 $\text{μl}$ of suspended cells and transfer to the 0.5 ml tube and then add 15 $\text{μl}$ of Trypan Blue and mix these well by re-pipetting. The cell to stain ratio must be 1:1.

2. To each well of the countess slide, add 10 $\text{μl}$ of the stained cells.

3. Measure the cell count using the Invitrogen Countess II as follows:

   • Insert each well into the slot.

   • Select Capture once the focus is completed.

   • Note down the cell counts for both wells in the lab notebook.

   • Take average cell count to get an estimate of how many cells are

     there in 1 ml of the suspension. Use this value to determine what volume of cells is required to be passaged into the new flask.

Seeding/Plating cells

1. According to the cell line, determine what would be the optimal

   seeding density and calculate the volume of cells needed to obtain that density.

NOTE 6: Do not seed at less than 20%-25% density as the cells will not be able to grow because of sparsity.

2. Put the respective volume of media in a new flask.

3. Add the calculated volume of cells into the new flask. Gently mix

   the cells in the media by grazing the flask back and forth on the work area surface to ensure even distribution.

4. View the flask under the microscope to ensure even distribution of

   cells.

5. Place the flask carefully in the primary incubator. Make sure to not

   rock it too much so as to avoid disturbing the even distribution of the cells.

6. Allow cells to grow for respective no. of days in the incubator at

   37$℃$ and 5% CO2.

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7. Disposals | Spills | Incidents

Waste (see waste segregations and collection SOP for complete information)

1. All waste containers must be washed thrice with a minimum 10% Bleach solution and discarded into the Bio-hazard dustbin next to the hood.

2. All waste containers should be properly labelled with the following information:

   • PI's name

   • User | Responsible person

   • Content

   • Waste class (e.g. chemical, biohazard, radioactive, etc.)

   • Waste type (e.g. sharps, plastic, glass, metal, etc.)

= Subculturing Adherent Cells (Cont.) =

3. All waste containers must be washed thrice with a minimum 10% Bleach solution and discarded into the Bio-hazard dustbin next to the hood.

4. All waste containers should be properly labelled with the following information:

   • PI's name

   • User | Responsible person

   • Content

   • Waste class (e.g. chemical, biohazard, radioactive, etc.)

   • Waste type (e.g. sharps, plastic, glass, metal, etc.)

   • Waste "status" (e.g. liquid, solid, etc.)

5. Discard bio-hazard glass materials in the Bio-hazard sharps disposal located on the tissue culture, next to the water bath.

Spills

All spills in the Tissue Culture Room must be cleaned immediately with 70% Ethanol and the used paper towels must be discarded into the Bio-hazard dustbin.

Incidents

1. External contamination

• Discard all reagents that were last used in the Tissue Culture Room.

  Ensure they are triple washed prior to disposal.

• Empty the hood as well the incubators and clean all their surfaces

  with 10% bleach solution.

• Make sure everything has been cleaned and wiped well prior to

  setting things back up.

2. Cross contamination

• Discard the respective flask.

• Clean all pipettes used.

• Thaw a fresh vial to start culturing the cells.

8. References

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1. Thermo Fisher standard protocol: https://www.thermofisher.com/us/en/home/references/gibco-cell-culture-basics/cell-culture-protocols/subculturing-adherent-cells.html

2. Useful numbers for cell culturing: https://www.thermofisher.com/us/en/home/references/gibco-cell-culture-basics/cell-culture-protocols/cell-culture-useful-numbers.html

= Subculturing Adherent Cells (Cont.) =

// Protocol Summary//

NOTE: This protocol summary should be used only as "side bench quick reference" and only once you're fully familiar with the full protocol above, including hazards and safety measures.

Preparation

1. Warm the reagents to 37$℃$ for roughly 20 minutes.

2. Sterilize the hood and all working containers with Ethanol and place them inside.

3. Check for growing cell's confluency under the microscope.

Washing

1. Discard old media.

2. Wash the flask with PBS buffer.

Dissociation/Trypsinization

1. Add 1 ml of trypsin to the flask.

2. Incubate for 2 minutes to promote dissociation.

Trypsin inactivation and removal

1. Add 3 ml of media and ensure all cells are washed off the flask's surface.

2. Transfer 4 ml of cell suspension into a 15 ml tube.

3. Centrifuge cells at 200 g for 5 minutes.

4. Discard supernatant and resuspend the cells in fresh 2 ml media.

Cell counting

1. Mix 15 $\text{μl}$ of cells with 15 $\text{μl}$ of trypan Blue stain in a 0.5 ml tube.

2. Add 10 $\text{μl}$ of stained cells to each of the two wells on the countess slide

3. Determine cell count using Invitrogen Countess II

Seeding/Plating cells

1. Determine seeding density with respect to the cell line and the experiments to be performed.

2. Add determined volume of cells to fresh media in a new flask and label it properly.

3. Incubate respectively at 37$℃$ and 5% CO2.