1. CELL CULTURE GLOSSARY
2. INTRODUCTION TO CELL BASED ASSAYS
3. REQUIREMENTS FOR ASSAY PROOF OF CONCEPT FOR CELL BASED ASSAYS
4. TISSUE CULTURE ASSAY DEVELOPMENT, OPTIMIZATION AND VALIDATION FLOW CHART
5. TYPES OF TISSUE CULTURE ASSAYS
   • Proliferation Assays
   • Apoptosis Assays
   • Gene Expression
   • Activation Assays
6. BATCH CO-TRANSFECTION AND CELL BANKING PROTOCOLS
7. CRYOPRESERVATION OF CELLS
8. CELL COUNTING PROTOCOLS
9. FACTORS FOR CELL BASED ASSAY DEVELOPMENT
10. TYPICAL CELL CULTURE PROTOCOL
11. STANDARD HTS CELL CULTURE PRACTICES TO REDUCE CONTAMINATION RISK
12. EXAMPLE, TRANSIENT TRANSFECTION OF CELLS
13. TRANSIENT TRANSFECTION METHODS
14. TRANSFECTION CATIONIC MATRIX REAGENTS
15. GENERAL CELL BIOLOGY INFORMATION
16. REFERENCES

Cell Culture – Establishment and maintenance of cultures derived from dispersed cells taken from original tissues, primary culture, or from a cell line or cell strain.

Cell Line – Immortalized cell, which have undergone transformation and can be passed indefinitely in culture.

Cell Strain – Cells which can be passed repeatedly but only for a limited number of passages.

Cell Clones – Individual cells separated from the population and allowed to grow.

Primary Culture – Cells resulting from the seeding of dissociated tissues, i.e. Huvec cells. Primary cultures often lose their phenotype and genotypes within several passages.

Cell Passage – The splitting (dilution) and subsequent redistribution of a monolayer or cell suspension into culture vessels containing fresh media.

Confluency – The confluency of a culture in a T flask or in a plate or dish is based on the amount of space between the cells. The confluency of the culture often influences the growth of the culture and expression.

Anchorage Dependent (Attached) Cells – Cells which require a substratum to divide and produce a monolayer.

Transient Transfection – The introduction of foreign DNA into a cell to allow the expression of the DNA into the host cell. Protocols are available for opening transient “holes”in the cell membranes allowing plasmids, or siRNA to enter the cell. Cells capable of being transfected or often referred to as “competent cells”. The DNA is not incorporated into the genome therefore, making the event transient referring to the transfection as a transient transfection.

Stable Cell Line – The selection of a stably transfected cell is where the transiently transfected cells are transfected with a co-expressed selection marker. Typical systems that exist include resistance to antibotics such as neomycin phosphotransferase, conferring resistance to G418, etc. The culturing of the cells can be done as a mixed population or by single cell culture to obtain cell clones from one single integration event.

Monolayer – A layer of cells one cell thick, grown in a culture.

Suspension Culture – Cells which do not require attachment to substratum to grow, i.e. anchorage independent. Cell culture derived from blood are typically grown in suspension. Cells can grow as single cells or clumps. To subculture the cultures which grow as single cells they can be diluted. However, the cultures containing clumps need to have the clumps disasociated prior to subculturing of the culture.

Density-Dependent Inhibition of Growth – Reduced response of cells upon reaching a threshold density. These Cells recognize the boundaries of neighbor cells upon confluence and respond, depending on growth patterns, by forming a monolayer. Usually these cells transit through the cell cycle at reduce rate (grow slower).

Differentiation – Property of cells to exhibit tissue-specific differentiated properties in culture.

Hatch – To bring cells out of the freezer; to start a culture from a freezer stock.

Thaw – Same as hatch.

Defreeze (Defroze) – Same as hatch.

Split – To subculture/passage cells; see cell passage.

Pass – See cell passage.

Carry – To maintain a cell line by subculturing in tissue culture medium containing nutrients that will maintain the phenotype and genotype of the cell line.

Plate – To aliquot cells into microtiter plates; plates can be 6, 12, 96, 384, or 1536 well; as opposed to dishes of either circular or rectangular shape, commonly a 500 cm2 culture dish.

1. Receptor of interest has been expressed in a suitable cell line and functional expression has been verified. Receptor expression has been verified by RT-PCR or Western blot.
2. Sample preparation has passed QC guidelines above.
3. Preliminary source for all reagents has been identified.
4. Early passage stable cell lines are available and free of mycoplasma contamination.
5. Transient transfection assays: appropriate cell line and transfection procedure demonstrated. Plasmids available and passes QC sequence and restriction mapping criteria (above).
6. Cell culture details are available as written SOP. This includes the number of cells (not dilution eg 1/10) used for passage, passage frequency, limit passage numbers for an assay, activity stability as a function of cell passage and density, and optimum cell density for target activity.
7. Biological activity (>90%) is target specific as demonstrated by transfection controls, comparison to parental cell lines, pharmacology, and/or tool compound activity.
8. Assay signal is dependant upon amount of cells present.
9. Assays enabled in DHT: Preliminary data showing a saturable activator and/or inhibitor response with sufficient signal window using a QB-supported assay format.

1. Thymidine uptake is measured by using 96 well plates coated with scintillant or using filter binding methods. Plates containing scintillant detect the thymidine taken up by the cells by using 14C or 3H.
2. WST reagents: A colorimetric assay for the quantification of cell proliferation and cell viability, based on the cleavage of the tetrazolium salt WST-1 by mitochondrial dehydrogenases in viable cells (a soluble version of MTT or MTX)
3. Alamar Blue™: A colorimetric assay
4. There are several non-radioactive, fluorescence-based assays that are not dependent on cellular metabolism. The fluorescent dye binds nucleic acids and the fluorescesences can then be measured quantitatively or qualitatively. Propidium Iodide, Hoechst or other live dyes can be added to cells. The fluorescence can than be detected using various high content imaging instruments. The cell number can then be quantitated based on the fluorescence. DNA content can also be quantitated using the tools available in the imaging instruments.
5. Chemiluminescent, non-separation assay kits for the determination of viable cell numbers are also available for either proliferation or cytotoxicity assays.

1. Annexin V staining
2. Tunnel staining
3. Caspase activity
4. Nuclear Fragmentation using microscopes, or other imaging platforms

1. Transient transfection of a gene of interest into a cell using various reporter systems. There are numerous transient transfection reagents and protocols available that have been optimized for various cell lines and promoters.
2. Stable transfection of a gene, is where the gene of interest is stably integrated into the cell. Stable transfections can take a lengthy period of time to be generated.

1. A. Measurements of intracellular second messengers: calcium flux, cAMP. There are numerous assay formats available for measuring cAMP including ELISA’s, radioactive formats, homogenous formats including FRET assay formats.and alpha screen.
2. Uptake of various cellular components measured by using radioactive ligands.
3. Activation of metabolic pathways:measurement of phosphorylation events intracellular or intranuclear.

1. Counting Cells for Transfection
   • Allow both the 0.05% Trypsin and the Assay media to warm up at room temperature prior to detaching cells.
   1. Remove the flask out of the incubator (37°C and 5% CO2) that will be used for counting.
   2. Aspirate off the media with an aspirating pipette attached to the vacuum source located within the culture hood.
   3. Add 10mls of Dulbecco’s PBS to each flask. Next, rock the flask(s) back and forth once making sure to wash the side of the flask with the cells attached.
   4. Aspirate off PBS.
   5. Add 3 ml of 0.05% Trypsin/flask. Rock the flask(s) back and forth to coat the cells with the Trypsin.
   6. Let sit at room temperature for 3 minutes. Whack flask to detach cells.
   7. Add 7mls media to flask to quench the Trypsin. Pipette up and down several times to break up clumps.
   8. Transfer the cell mixture to a 50 ml blue-top centrifuge tube and mix well.
   9. Count the cells three times using a Coulter cell counter and calculate the average.
   10. Next, calculate the number of cells using the following formula:

       (Avg. #of counts)/(vol. the counter will count, µl) X (vol. of isoton, µl )÷(vol. of the sample, µl) X (total vol. of the cells sampled from, µl)
       Example: 6279 counts/500 µl X 20,000 µl ÷ 100 µl X 10,000 µl = 25.11 million

2. Transfections
   1. Prepare the Serum-free media and transfection reagent mix in a 250 ml orange-top conical centrifuge tube according to the optimized ratio for transfection. Mix gently halfway through and after adding all of the reagent and incubate for 5 minutes. DO NOT touch transfection reagent to the plastic sides of the tube. Dispense directly into the SFM.
   2. Add appropriate amounts of DNA to the tube with the SFM and transfection reagent mix. Mix, by tapping the tube gently after additions.
   3. Incubate for 30 minutes at room temperature.
   4. During this incubation period, aspirate media from the T225 flasks to be transfected and add back 38mls of Assay Media. Place back in the incubator at 37°C, 5% CO2.
3. Cell Banking
   • Allow both the 0.05% Trypsin and the Assay media to warm up at room temperature prior to detaching cells.
   1. Remove the flasks out of the incubator (37°C and 5% CO2).
   2. Aspirate off the media with an aspirating pipette attached to the vacuum source located within the culture hood.
   3. Add 10mls of Dulbecco’s PBS to each flask. Next, rock the flask(s) back and forth once making sure to wash the side of the flask with the cells attached.
   4. Aspirate off PBS.
   5. Add 3 ml of 0.05% trypsin/flask. Rock the flask(s) back and forth to coat the cells with the Trypsin.
   6. Let sit at room temperature for 3 minutes. Whack flask to detach cells.
   7. Add 7mls media to flask to quench the trypsin. Pipette up and down several times to break up clumps.
   8. Transfer the cell mixture to a 250 ml orange-top centrifuge tube and mix well.
   9. Divide cell suspension between orange-top tubes.
   10. Count the cells three times using a Coulter cell counter and calculate the average.
   11. Next, calculate the number of cells using the following formula:

       (Avg. #of counts)/(vol. the counter will count, µl) X (vol. of isoton, µl )÷(vol. of the sample, µl) X (total vol. of the cells sampled from, µl)
       Example: 6279 counts/500 µl X 20,000 µl ÷ 100 µl X 10,000 µl = 25.11 million

   12. Spin tubes in centrifuge at 1500 RPM’s for 5 minutes.
   13. Remove supernatant.
   14. Divide total number of cells (see #9) by 50 (cell concentration/ vial frozen: 50X10e6) = mls freezing solution to add to cell pellet.
   15. Pipette up and down several times to break up cell clumps.
   16. Aliquot 1 ml of cell suspension to a 2 ml cryogenic vial.
   17. Place vials in a Mr. Frosty and place in -80°C freezer for approximately 4 hours.
   18. Remove and place in Liquid Nitrogen tank for long term storage.

       Freezing Solution

       • 90% Fetal Bovine Serum (Charcoal Stripped FBS)
       • 10% DMSO
4. Validation of Frozen Cells
   1. Remove vial from liquid nitrogen storage.
   2. Thaw quickly in 37°C water bath.
   3. Bring up slowly to total volume of 20-30 mls of cold Assay media.
   4. Spin at 1500 RPM’s for 5 minutes. Remove supernatant.
   5. Re-suspend in 10 mls assay media. Take a 100 ul aliquot of cell suspension and add to 20 mls Isoflow.
   6. Count the cells three times using a Coulter cell counter and calculate the average cell count.

      (Avg. #of counts)/ (vol. the counter will count, µl) X (vol. of isotonic, µl) ÷(vol. of the sample, µl) X (total vol. of the cells sampled from, µl )
      Example: 6279 counts/500 µl X 20,000 µl ÷ 100 µl X 10,000 µl = 25.11 million

   7. Seed a 96 well or 384 well microtiter plate following one of the below equations to determine cell number in suspension.

      384 well format (30,000 cells in 50 ul):

      1. (11.5 mill. Cells/plate) (_____plates, included 2 extra) =_____mill. Cells needed
      2. (____mill. Cells needed for assay accounting for extra) / (____mill. cells/ml) = ______ml of cells from cell stock
      3. (____mill. cells/ml) (____ml of cells from cell stock) / (0.6 mill. cells/ml) = ml total
      4. (____ml total) – (____ml of cells from cell stock) = ml of media

      96 well format (50,000 cells in 80 ul):

      1. (4.8 mill. Cells/plate) (____plates, included 1 extra) = ____mill. Cells needed
      2. (____mill. Cells needed for assay accounting for extra) / (____mill. cells/ml) = ____ml of cells from cell stock
      3. (____mill. cells/ml) (____ml of cells from cell stock) / (0.625) mill. cells/ml) = ml total
      4. (____ml total) – (____ml of cells from cell stock) = ml of media
   8. Incubate overnight overnight in 37°C, 5% CO2
   9. Dose with appropriate dose response curve for each receptor

1. Cryopreservation
   1. Remove media from flask.
   2. Wash cells gently with 10mls D-PBS and aspirate.
   3. Add 3mls Trypsin-EDTA , let sit on cells for 2-3 minutes.
   4. Tap flasks to detach cells.
   5. Add 7mls of Growth Media (see recipe) to flask. Pipet up and down several times and transfer to a 50ml conical centrifuge tube. Take a 100ul aliquot of the cell suspension and add to 20mls Isoflow/Isoton and count using Coulter Counter. Count cells three times.

      (Avg. #of counts)/(vol. the counter will count, µl) X (vol. of isoton, µl )÷(vol. of the sample, µl) X (total vol. of the cells sampled from, µl)
      e.g. 6279 counts/500 µl X 20,000 µl ÷ 100 µl X 10,000 µl = 25.11 million

      Divide by the number of cells you want to freeze down and add that final number in mls of freezing solution.

      Example: 25.11 million ÷ 5 million = 5.022 (number of mls of freezing solution to add to get 5 X 10e6 cells per vial)

   6. Spin cells in centrifuge at 1500rpms for 3 minutes.
   7. Remove supernatant and resuspend cells in freezing medium (see below) @ a concentration of 5 X 10e6 cells per ml in a 1.5ml Cryo vial.
   8. Label vials with cell line, passage #, freeze down date, notebook number if possible and number of cells frozen.
   9. Place in a freezing container and place in a -80°C freezer for ~ 2 to 4 hours.
   10. Remove and place in liquid nitrogen tank for long term storage.
       
       Freezing Solution
       • 10% DMSO + 90% Characterized FBS (You want to use the FBS used in the Growth media for making up this solution)

2. Thawing of Cryopreserved Cells
   Centrifugation Method:
   1. Remove cells from liquid nitrogen storage and thaw quickly in a 37°C water bath.
   2. Remove cells from cryo vial and place in 50ml conical tube.
   3. Add ~20 to 30mls of cool Growth medium slowly to the tube.
   4. Centrifuge cells @ 1500rpms for 5 minutes.
   5. Discard supernatant.
   6. Resuspend cells in Growth media and count cells and seed flasks.
   Direct Plating Method:
   1. Remove cells from liquid nitrogen storage and thaw quickly in a 37°C water bath.
   2. Plate cells directly with Growth medium. Use 15-20mls of Growth Medium/ 1ml frozen cells @ 3 X 10e5.
   3. Culture cells 6-8 hours. Replace with fresh Growth Medium to remove the cryopreservative.

Factor	Range/Rationale
Cell type	Primary cells vs. an established cell line Primary cells are often difficult to culture. Phenotype and viability may change within a few passages. Established cell lines are typically easier to culture and usually maintain viability and phenotype over numerous passages.
Number of cells per well	Cell density will depend on the cell type and the type of assay being performed. Start with a broad range of cell concentrations and then narrow down the optimal cell number per well using experimental design
Cell passage number	Cells can change their responsiveness over passage number. Primary cells in particular have a tendency to change over passage number. Receptor number may be dependent on passage number or condition that the cells are maintained under.
Growth medium	Each cell type has specific medium optimized for growth. This information can be found in ATCC or in publications.
Growth serum type	Serum type is usually specified in the literature or in ATCC. Dextran Charcoal (DCC) treated serum removes proteins, which may interfere with the assay. Cells may require Horse serum in addition to Fetal bovine serum.
Growth serum concentration	Serum is almost always required for growth conditions unless the medium has been optimized for serum free conditions. Ranges are from 1-15% serum concentrations. Serum free conditions are often used to prevent interference with compounds binding to protein or to increase the stimulation of the inducer.
Treatment medium	Medium being used during the treatment can differ from that used in growth medium.
Treatment serum type	Serum type is usually specified in the literature, by ATCC, or from the originator of the cell line. However, compound testing can be done in serum free conditions.
Treatment serum concentration	Serum free conditions are often used during treatment of compounds once cells have been established in serum to remove effects of serum. Low concentrations of BSA are often substituted to prevent non-specific protein binding from occurring.
Incubation time for pretreatment	Dependent on response desired for assay and biological relevance.
Treatment incubation time	Dependent on desired assay response and biological relevance.
Plate type	Plate type is assay dependent. Fluorescent assay: black or white; Luminescent: white or black; Radioactive: Plates containing scintillant coated on the bottom or on the bottom and sides, or filter plates for filter binding. Colorimetric: clear tissue culture treated plates for assays measuring proteins in a cell culture supernatant like an ELISA, the supernatant is transferred to the ELISA plate.
Coating type	Some cells adhere better with some type of coating, reducing variability and enhancing the response. The coating can be poly-D-lysine, fibronectin, collagen or other matrix, that enhances cell attachment to the plate.
pH	Neutral
Inducer/Inhibitor concentration	Dependent on experimental conditions.
Radioactive tracer concentration	Based on optimal signal window obtained during assay optimization.
Addition time of radioactive tracer	Based on optimal signal window obtained during assay optimization and biological relevance. The simplest method is to add the radioactive tracer at the same time as the inducer and then measure the response over a period of time post induction.
Type of radiation	14C is usually needed for plates containing scintillant coated on the bottom to get an optimal signal window. Other types of radioactive tracers may be used for other assay platforms.

1. Place media and trypsin in water bath (37°C).
2. Take flask of cells out of incubator.
3. Examine cells under microscope to determine health/condition.
4. In hood, remove medium from flask by aspiration.
5. Wash cells once with 5-10 ml of Phosphate Buffered Saline.
6. Aspirate off PBS.
7. Add 5-10ml trypsin or dissociation solution to cells.
8. Rock flask to allow above to cover cells.
9. Incubate 2-5 minutes or until cells begin sloughing off the flask.
10. Tap flask to see if cells have released from bottom of flask.
• If not, incubate further 1-2 minutes or place flask in 37° incubator for few more minutes.
• If so, go to 11.
11. Add equal volume of medium to cells.
12. Pipet up and down several times to break up cell clumps.
13. Pool all cell into one container.
14. Take out 1ml sample to count.
15. Count on hemacytometer, or automated cell counters like a Coulter Counter or Vi-CELL.
16. Do calculations to determine cell density.
17. Calculate amount of pool needed for desired cell number for project.
18. Take off that amount of cells and centrifuge at 1000rpm for 5+minutes. Determine amount media to resuspend cell pellet in.
19. Resuspend pellet in appropriate amount of medium.
20. Seed or plate as needed. Automated dispensers for cell plating will decrease variablility of cell seeding in 96, 384 or 1536 plates.

• When moving things (media bottles, pipet tips, etc.) into a biosafety cabinet (hood) you should wipe them down with 70% EtOH. Be sure to cover the entire object.
• Wipe out hoods before and after use with 70% EtOH. UV weekly. Lysol (3%) weekly.
• Hoods should be completely cleaned at least 3 times a year. This entails taking the surface tray and grills out, washing them and the area beneath them with Lysol then EtOH, and removing any debris found below the tray. Autoclaving the tray and grill are acceptable.
• Bleach vacuum flasks and lines. Change the flask weekly even if not full.
• Wipe out incubators at least once a month with Lysol followed by 70% EtOH. This is a known source of fungal contamination. When opening an incubator, check for fungal growth on the shelves and around the seals.
• Empty biowaste containers regularly, preferably at least twice a week. All waste should be double bagged (bag into container then another bag inside the first).
• Do not carry large stacks of plates or flasks unless you use a cart.
• Wear gloves. Make sure your lab coat is not grimy.
• Bleach any container that has contained cells for a few minutes. The bleached media can be washed down the sink. However, do not open CONTAMINATED containers in the main lab area. If you have a contamination, autoclave it BEFORE opening.
• Fluid delivery lines/ drain lines should be rinsed with 70% EtOH chased with dH2O every day after use. This is a known source of contamination. This would include multidrop heads, multimek lines, MRD8 lines, etc. . If dispensing media with a multidrop, rinse head then autoclave the head. You should keep an autoclaved head in reserve if possible in case of failure of the daily one. If using the Multimek to aspirate or plate cells, rinse the lines and wash station then autoclave the wash station. The autoclaving does not apply to heads used for 384 well delivery
• If you have a contamination event, DO NOT OPEN IT IN THE MAIN LAB! Contact your supervisor or a cell culture person to help in identifying the contamination and the source. Also, make others using cells aware that you had a contamination event.
• Be careful not to touch pipets, media bottle openings, etc. . Touch events are a leading source of contamination in cell culture. If you do happen to touch a pipet discard it and get a new one. If you touch a bottle opening, wipe it immediately with an EtOH swipe and then filter that media through a 0.2um filter apparatus.
• Cleanliness is next to Godliness especially in cell culture. Keep your hood free of unnecessary clutter. Wipe up spills promptly. Try not to sneeze inside your hood. Drips should be promptly cleaned up with EtOH.

Note the sash level limit on your hood. There should be a mark or arrow on one side of the sash (glass) to tell you how high not to go with the sash. Too high will disrupt the air flow and compromise your hoods sterility.

The Lipofection protocol is a relatively simple method that has been used for high throughput screening. Various lipid and matrix reagents can be compared for using the Lipofectamine protocol and measuring the transfection of enhanced green fluorescent protein (EGFP) and β-galactosidase (β-gal) marker plasmids to determine optimal reagents. Fugene 6 has been observed to work best in the presence of serum and resulting in little or no toxicity. Following is a list of transfection cationic matrix reagents. Measurement of the efficiency and toxicity of the transfection can be used to determine the optimal transfection cationic matrix reagent.

Once the optimal transient transfection method and cationic matrix reagent have been selected then additional factors can be considered in developing a method for optimizing a transient transfection protocol.

Day	Protocol	Variables
1	Seed cells	Cell number is dependent on cell type Type of culture flask Optimal range is 50% density on day 2 (40-60%)
2	Transfect cells	Protocol of transfection is dependent on reagents used (see list above on transfection reagents) Variables to be tested: DNA amount, Reagent amount, DNA: Reagent ratio, serum concentration, medium type, time of transfection. These variables are optimized according to cell type.
3,4 or 5	Determine transfection efficiency, end product or reporter expression Induction or stimulation	Depending on the cell type the assay for gene expression is usually measured between 24 to 72 hours after transfection. Induction or stimulation is usually done 48 hours after transfection, and then determine expression levels. Method for determining expression will also depend on type of expression- GFP can be monitored by visual inspection; luciferase is measured by cell lysis, addition of substrate and measurement of light output.

1. http://www.atcc.org/
2. http://www.protocol-online.org/prot/Cell_Biology/index.html
3. http://www.protocol-online.net/cellbio/cell_culture/cell_culture.htm - Nice sight with cell culture protocols, etc.
4. http://www.cellsalive.com/
5. http://www.tissue-cell-culture.com/docs/libary/tc_trouble.html - This site has excellent list of cell culture trouble shooting
6. http://www.answers.com
7. http://en.wikipedia.org/wiki/coulter_counter
8. http://www.schuett-abraham.de/glossar-en.htm
9. http://www.invitrogen.com/tissue_culture.htm
10. http://www.amaxa.com/stable-transfection.html
11. http://www.biochemweb.org/apoptosis.shtml
12. http://www.caspases.org/