Cell Culture Protocols and Notes

1.0Preparing Necessary Reagents

1.1Media Components and Other Necessary Solutions

1.1.1Heat inactivated fetal bovine serum (FBS)

We purchase heat inactivated fetal bovine serum which should be sterilized and tested for mycoplasm contamination. This solution will arrive frozen on dry ice. Thaw the solution in the water bath and then separate into 50mL aliquots (large falcon tubes). The aliquots can be refrozen.

1.1.2Penicillin streptomycin

We purchase the penicillin streptomycin from Sigma. This antibiotic is used to prevent bacterial growth in the media. Penicillin-G prevents the final formation of the bacterial cell wall. Streptomycin binds to the 30S subunit and cause misreading in the of bacterial DNA. The solution will arrive frozen on dry ice. Thaw solution in the water bath and then separate into 5mL aliquots (small falcon tubes). The aliquots may be refrozen until needed. The stock solution (P0781 Sigma Aldrich) contains 10,000 units of penicillin-G, and 10mg of streptomycin per mL. The final concentration in our media is ~ 100U/mL penicillin and 100ug/mL streptomycin.

1.1.3L-glutamine

L-glutamine is an essential amino acid required for most if not all cell culture media formulations. The glutamine serves as an energy source for rapidly diving cells, and those which must synthesize large quantities of nucleic acids and proteins. L-glutamine should be in the DMEM which we order, however it does degrade over a period of 2-6 weeks at 4ºC, and with exposure to light. If you are not going through media very fast it will be necessary to supplement your media with fresh L-glutamine after about 4 weeks, or if you note a large change in the rate of growth of your cells. Ammonia is a byproduct of the L-glutamine degradation, and old media should be discarded (> 6 weeks) to avoid harming the cells.

1.1.4Non-essential amino acids (NEAA)

Non-essential amino acids are added to media to increase the nutrients available to the cells. The stock solution is very acidic (pH 1.5 – 1.7), and thus if added to a non-buffered solution you may want to check the pH to ensure it is not too acidic. The solution contains glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline and L-serine.

1.1.5HEPES

HEPES is used to keep the pH stable between 7.2 – 7.6. We add HEPES to media which is used for imaging or when cells are removed from the incubator for extended periods of time. Excessive cellular metabolism or rapidly growing cultures may acidify quickly and small amounts of HEPES can help stabilize the cells.

1.1.60.25% Trypsin solution

Stock solutions of trypsin are purchased in 2.5% strength, and kept in the freezer. To prepare working solutions we typically add 1.0 mL of the trypsin to 9.0mL of PBS (-) (contains no Ca2+/Mg2+). These solutions can be prepared in the small 15mL falcon tubes and placed in the freezer. When needed thaw the solution in the water bath.

1.1.7Citric saline solution

Some cell lines are much less adherent than the CHO cells, and thus require less coaxing in order to passage them. Citric saline solution works well for HEK293T and AD293 cell lines. We begin by first making a 10X stock solution of citric saline and then diluting to a 1X working solution. The following recipe is for 500mL of the 10X solution:

1.35MKCl (MW 74.5 g/mol  50 g)

0.15Msodium citrate (294 g/mol for trisodium salt  22g)

Dissolve the above in 500mL of pure Millipore water. Sterilize the solution by autoclaving. The stock solution can be kept for up to 3 months at room temperature. Dilute this solution by placing 5mL of the 10X solution in 45mL of pure sterile water. This solution can be kept in the fridge.

1.1.890% FBS/10% DMSO freezing media

Prepare this solution by thawing a 50mL aliquot of the FBS. Remove 5.0mL of the media, and replace it with 5.0mL of DMSO. We use DMSO to prevent ice crystal formation, which would result in damage to the cells during the freezing process. Caution: DMSO can be toxic to some cells lines, it is also possible to use 10% glycerol in these cases.

1.1.94% paraformaldehyde

This solution is used to fix cells for imaging purposes. The 4% refers to 4g per 100mL of pure water. The mixture will be very cloudy, heat the solution carefully on a low setting (3) with the cap loose swirling occasionally until all the powder has dissolved. Take care not to breath in fumes as they are toxic. This solution should also be kept frozen. When preparing paraformaldehyde avoid breathing in the powder, wear protective gloves, lab coat, safety goggles.

1.1.10Geneticin (G418 Sulfate)

Geneticin is an aminoglycoside antiobotic which is toxic to yeast, bacteria, protazoans and mammalian cells. It is possible to introduce a genetic resistance to cells (on transposons Tn601(903) and Tn5). The antibiotic interferes the function of the 80S ribosomes and disrupts protein synthesis in non-resistant cells. According to some sources potency is achieved at concentrations > 450 g/mL, these higher concentrations can be used for selection, while lower concentrations around 200 g/mL are suitable for maintenance of an already stable cell line. For our CHO (+) media we add 0.235g of the Geneticin to the media yielding a total concentration of 370g/mL.

1.1.110.1M Bicarbonate Buffer

This buffer is used in for reactions between succinimidyl esters and amines forming amide bonds. The basic pH of this buffer is used to keep the -amino group in the non-protonated form during the reaction. You can not use buffers which contain amine groups (ie. TRIS) for this type of reaction because the buffer itself will compete with the amine and react with the activated dye.

The following recipe will yield you a 0.2M bicarbonate buffer with a pH ~9.2. Depending on the type of ester you will need to adjust the pH using the pH meter and the 1.0M HCl solution.

Obtain sodium carbonate (Na2CO3) and sodium bicarbonate (NaHCO3) from the shelf. You will actually prepare two different stock solutions and them mix them together to obtain the buffer. It is wise to make extra stock solution so that you do not have to do this step each time.

Preparation of the 0.2M Na2CO3 stock solution:

MW = 105.99 g/mol

Vol = 0.500 L

C = 0.2 mol/L

n = 0.1 moles

mass needed = 10.56 g

Preparation of the 0.2M NaHCO3 stock solution:

MW = 84.11 g/mol

Vol = 0.500 L

C = 0.2 mol/L

n = 0.1 moles

mass needed = 8.41 g

Preparation of final buffer mixture:

Mix 92.0 mL of NaHCO3 with 8.0 mL of Na2CO3 to yield a final volume of 100.0 mL.

Adjust the pH using HCl to obtain the desired pH level. This solution should be made fresh every month.

1.1.12LB Broth for Maxi Preps

We purchase LB broth powder in small pre-weighed packages. Each package is suitable for preparing 500mL of media. Each maxi prep requires about 300mL of LB broth. Once prepared sterilize the media using the programmed “liquids” setting on the autoclave leaving the cap slightly un-screwed. Once autoclaved media can be stored at room temperature for up to 3 months, as long as it remains un-opened.

1.1.13X

1.2Media for Cell Culture

We are now using washable and reusable glass bottles to prepare our media and our PBS solutions. The media bottles have tops which are compatible with the disposable sterile filters from VWR. These bottles should be washed with bleach and water (3x with bleach and 9x with water), then dried and autoclaved. After being autoclaved you can spray the outside with EtOH and bring into the laminar flow cabinet. I would recommend opening the bottle and placing the lid upwards on the bottom of the cabinet and turn on the UV light for 10 minutes just to be sure.

1.2.1CHO K1 (-) Media

This media is suitable for non-transfected CHO K1, or transiently transfected CHO cells which do not have any antibiotic resistance.

To prepare the media will need the following:

  1. 430mL of DMEM
  2. 50mL of heat inactivated FBS
  3. 5mL of Pen/Strep
  4. 5mL of Non-essential amino acids
  5. 10mL of L-glutamine

Mix all reagents together and run through a sterile filter.

1.2.2CHO K1 (+) Media

This media is suitable for CHO K1 cells which stably express fluorescent proteins and are resistant to geneticin.

To prepare the media you will need the following:

  1. 400 mL of DMEM – Low Glucose
  2. 50 mL of FBS
  3. 5 mL of Pen/Strep
  4. 5 mL non-essential amino acids
  5. 10 mL of L-glutamine
  6. 0.235g of geneticin

The geneticin is not immediately soluble in the DMEM. It is recommended that you add the geneticin first to the warm DMEM to increase the solubility and maximize the concentration of geneticin in the sample.

1.2.3AD293T Media

These cells are variants of HEK 293 cells which are slightly more adherent. To prepare this media you will need the following:

  1. 445 mL DMEM - High Glucose
  2. 50 mL FBS
  3. 5 mL Pen-Strep

If this media is for the AD291 NK1 cells which stably express the NK1 receptor reduce the amount of DMEM by 3 mL and add 3 mL of G418 antibiotic.

If we do not have high glucose media you can supplement low glucose media with 1.750g of D-glucose to achieve the necessary amount of glucose.

1.2.4HEK293 Media

To prepare this media you will need the following:

  1. 445 mL DMEM – High glucose
  2. 50 mL FBS
  3. 5 mL Pen-Strep

If we do not have high glucose media on hand you can supplement low glucose media with 1.750g of D-glucose to achieve the necessary amount of glucose.

1.2.5HeLa Media

HeLa media is the same as HEK293 media described above. HeLa cells can be easily contaminated, and have been shown to contaminate other cell lines. For this reason it is advised that if you have both HEK293T and HeLa cells going, separate the media into two separate bottles to avoid cross-contamination of the cell lines. It is also possible to use CHO(-) media with the HeLa cells again, be very careful to avoid contamination of the HeLa cells

1.3Imaging Medias

Growth media contains phenol red as an indicator allowing us to observe large changes in the pH of the media. Phenol red does have both substantial autofluorescence and can act as a screen preventing the fluorescence from other proteins from being observed. This is less problematic when examining the basal membrane of adherent cells using an inverted microscope, but in general it is good practice to image the cells in media which is phenol red free (-). Because we often remove the cells from the tightly controlled CO2 environment we want to prevent the buildup of toxic metabolites which will limit the life of the cells for imaging. We do this by adding HEPES to the buffer at low concentrations (~ 20 – 25 mM). We often add radical scavenger such as ascorbic acid to help reduce general phototoxicity. And finally, imaging media will often be FBS (-) free. Cells are often more responsive to stimulation with drugs when they have been deprived some FBS for at least 1 hour (but up to 12 hours is often ideal). This is particularly true for the EGFR containing cells. In order to get a nice dispersion of EGFR on the membrane we will starve the cells (of FBS) for 12 hours prior to imaging.

1.3.1For CHO K1 EGFR-GFP cells

EGFR can cluster in response to light stimulation. The addition of N-acetyl-L-cystein (NAC) can prevent this from occurring. However we have recently discovered that for transactivation studies this component shuts off the necessary pathways and thus for transactivation work you can omit the NAC.The imaging media is made up of the following components.

The HEPES and other sterile solutions can be aliquoted into 1 large falcon tube (50mL) in the laminar flow hood and then added to the Hanks Balanced Salts Solution. The media can be prepared outside the laminar flow cabinet because you will need to adjust the pH to 7.4. Once the pH has been adjusted use a filter and clean media bottle to sterilize the media.

  1. 400 mL of Hanks Balanced Salts Solution
  2. 10.6 mL of HEPES
  3. 5.0 mL of PenStrep
  4. 5.0 mL of MEM NEAA
  5. 0.2125g ascorbic acid (shelf)
  6. 0.3329g N-acetyl-L-cysteine (fridge)

1.3.2For AD293/HEK cells

We can prepare an imaging media from phenol red free (-) high glucose media. For imaging purposes we once again omit the FBS, but will add the Pen-strep to prevent bacterial growth. Importantly, we will have to add HEPES buffer.

  1. 400 mL of phenol red free high glucose media
  2. 10.6 mL of HEPES
  3. 5.0 mL of PenStrep
  4. 5.0 mL of MEM NEAA
  5. 0.2125 g ascorbic acid
  6. 0.3329 g N-acetyl-L-cysteine

2.0Thawing

Cells are typically frozen in 90% FBS and 10% DMSO freezing media. When cells are removed from either the -80 or the N2(l) freezer you must thaw the cells quickly. In order to prevent damage to the cells it is wise to place 8.5 mL of the appropriate media (warmed to 37ºC) in a 15 mL falcon tube. Thaw the cells at 37ºC in the water bath until about ½ of the sample has melted. Wipe down the vial with ethanol and add to the 8.5 mL of media. Place the cap on the falcon tube and invert to mix. Now there are one of two options.

1)Place the tube in the centrifuge and spin down for 5 minutes on setting 3. Suck out the media and re-suspend the cells in 10 mL of the appropriate media. Divide the 10mL of suspended cell solution into two small culture flasks. Make sure to label the flask with the passage number, date and cell line.

2)Divide the 10mL of suspended cell solution into two culture flasks. Make sure the label the flask with the passage number, date and cell line. Place flasks in the incubator.

Notes about these options: Some people are very concerned that the DMSO can harm the cells, and thus spinning down the cells and removing the media helps the cells recover from freezing faster. It has been my experience that the second method actually works better for our cells.

After 24 hours check the dishes to see whether the cells have become adherent. If there is a substantial amount of dead cells in the flask it is a good idea to switch the media to avoid poisoning the cells which are viable. The cells can take up 1 week to recover from the thawing process. Depending on how fast the cells grow you may need to passage the new cultures in a few days, or you may simply need to keep changing the media every few days. Changing the media helps ensure that while the cells are growing and dividing there is sufficient nutrients, and limited build up of toxic metabolites in the media.

3.0Freezing

Cells should be frozen when at low passage number, healthy and confluent. When you thaw cells check to see how many tubes we have of that cell line, if we have less than 2 tubes you must freeze new cells as soon as possible.

To freeze we grow the cells up in the large 75cm2 culture flasks. During normal passaging we add 5mL of freshly diluted cells to the flask and add 20mL of the appropriate media. Monitor the cells and freeze the cells when they are ~80 – 90% confluent. Some protocols suggest changing the media about X hours before you wish to freeze to ensure all the cells are in the X phase of growth. Once the cells are ready for freezing use the following protocol.

  1. Warm PBS (-), appropriate media and freezing media in water bath.
  2. Pre-label 5 tubes with the following information:
  3. Cell line and any expressing proteins (ie. CHO K1 EGFR-GFP)
  4. Passage number
  5. Date
  6. Have waiting a small cooler full of dry ice. Place an empty styrofoam 15 mL falcon tube holder in the dry ice and pack dry ice around the styrofoam. This will create a way for the cells to freeze slowly and uniformly over a 1 hour period. Some people suggest flash freezing the cells, but we find this method is suitable for preparing the cells for nitrogen freezing that same day.
  7. Remove the media from the flask using suction.
  8. Rinse with ~ 5 mL of PBS (-).
  9. Add about 1 mL of trypsin enough to cover the whole bottom of the dish. Remove half of the trypsin immediately and place the cells in the incubator for 5 minutes until they detach. For HEK or AD293 cells use citric saline, and do not place the cells in the incubator.
  10. Add 10mL of appropriate media to suspend cells. Using the 10mL pipette transfer all the suspended cells into a small 15mL falcon tube.
  11. Centrifuge at setting 3 for 5 minutes.
  12. There should be a large palette at the bottom of the tube.
  13. Suck up 9mL of the freezing media and add it to the tube forcefully enough to dislodge the pellet at the bottom. Mix well by sucking up and dispensing the solution a few times.
  14. Add 1.8mL of the suspended cells in freezing media to each of the labeled Nunc cryotubes. Place the cap on tightly.
  15. Place each tube in the empty styrofoam holder in the cooler of dry ice.
  16. Allow to freeze until solid. This should take between 1 – 2 hours.
  17. Once frozen the cells can be stored in N2(l). To do this you must first wrap each of the tubes in cryotubing.
  18. Prepare the cryotubing by cutting the long tubing into appropriate lengths and sealing one end.

Diagram: Cutting the cryotubing

Diagram: Sealing one end of the cryotubing