HCT-116 InstaCell Proliferation Assay with XTT - CE-111

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1 HCT-116 InstaCell Proliferation Assay with XTT - CE-111 Background With InstaCell Proliferation Assays you gain a tool that will help you to increase the efficacy in your screenings. The ready-touse assay releases you from all kind of preparative cell culture and enables a flexible scheduling. Cells are directly frozen in microwell plates and can be stored at -80 C for several months without losing viability. By adding medium, the cells are activated and can be instantly used for a cell based assay. You simply take out the needed quantity of plates from your freezer. Unnecessary handling of cells that might be a source of error is avoided. This makes InstaCell Proliferation Assays very convenient to use and increases the reliability of the assay. The HCT-116 InstaCell Proliferation Assay has been developed to screen for new substances that have an antiproliferative or cytostatic effect against tumor cells. The HCT-116 InstaCell Proliferation Assay (Cat. # CE-111) is provided as a kit including XTT. XTT is metabolized by living cells so total metabolic activity of the cells in the well is used to measure cell proliferation. XTT can be detected by absorbance and no extraction is necessary. PLEASE READ ENTIRE BOOKLET BEFORE PROCEEDING WITH THE ASSAY. CAREFULLY NOTE THE HANDLING AND STORAGE CONDITIONS OF EACH KIT COMPONENT. PLEASE CONTACT BIOMOL TECHNICAL SERVICES FOR ASSISTANCE IF NECESSARY. Components of CE-111 Kit Components and Storage Conditions KI-324 Multiwell Plate with Frozen HCT-116 Cells Storage: -80 C Use immediately after thawing. Do not refreeze. Quantity: 4x 96well plate KI-313 Culture Medium Quantity: 1x 50 ml KI-329 Dilution Buffer Quantity: 1x 50 ml KI-321 XTT Solution Quantity: 1x 25 ml KI-316 Positive control (5-Fluorouracil) Quantity: 2x 1.8 ml InstaCell Proliferation Assays are delivered on dry ice. Assay plate and reagents are stable for 6 months from the day of production. For the expiry date refer to the labels on the package. Other Materials Required A CO 2 -incubator (37 C, 5% CO 2 ) with humidified atmosphere (approx. 95%) is recommended. Alternatively, a standard incubator at 37 C might be used. In this case place the plates into a closed box together with a wet towel to avoid evaporation of the culture medium. Microplate-Reader with filter sets for: XTT (absorbance): 450 nm (reference above 650 nm). 1

2 Safety Information The HCT-116 InstaCell Proliferation Assay has been developed for in vitro research only. The assays must not be used in human or for any diagnostic, medical or drug purpose. All assay components of the kit have to be handled as potentially hazardous. Wear disposable glove and protective clothes while using the HCT-116 InstaCell Proliferation Assay. 5-Fluorouracil (5-FU) is harmful. It is irritating to eyes, respiratory system and skin. Do not breathe dust. The HCT-116 InstaCell Proliferation Assay contains living cells. The cell line is well characterized and has not been genetically modified. Inactivate cells by autoclaving for 20 min. at 121 C or incubate at ph >12 for 30 min before disposal. The purchaser of the assay is responsible for professional use of the product. BIOMOL International is not liable for any damage that results from transportation, storage, use, nonuse or disposal of the product. Overview of the Protocol Principle of the Assay The HCT-116 InstaCell Proliferation Assay has been developed to determine the antiproliferative effect of substances against human tumor cell lines. To avoid false positive results by contact inhibition of the cells, the seeding density has been carefully adjusted. The cells will not become confluent until after 96 hours of incubation. The assay plates are frozen and can be maintained at -80 C. After thawing, the cells can be activated by adding cell culture medium. No washing of the cells is necessary. After the cells have attached they can be incubated with the test samples. Finally a dye is added to quantify the proliferation of the cells. XTT can be used for the determination of cell mass. It passively penetrates the cells and is metabolized by mitochondrial and cytosolic enzymes. Yellow XTT is reduced to an orange colored formazan salt. XTT is soluble and diffuses passively into the surrounding medium. So, no extraction of the cells is necessary and the dye can be directly quantified in the cell culture medium. The intensity of the signal is proportional to the number of living cells and therefore a measure of their proliferation. From the data acquired for serial dilutions of an experimental sample, the IC 50 value can be calculated. The IC 50 describes the concentration of the experimental sample in which the metabolic activity of the cells, i.e. the proliferation, is reduced by 50% of an untreated control. IC 50 values can be calculated by non-linear regression or estimated graphically (Figure 1). Fig.1: Typical plot of InstaCell Proliferation Assay data as the percentage of living cells (negative control = 100%) against the concentration of the sample logarithmic scale. 2

3 Configuration of Assay Plates Depending on results that are expected from the assay, different test compound configurations can be used. It is important to carefully plan the configuration of an assay before starting. For valid results, two concentrations of the test compound should have no effect on the cells, two concentrations should completely inhibit proliferation of the cells and at least one concentration should lie on the slope between both extremes. If the range of effective concentration is known for a set of experimental compounds, small dilution steps (1:2) might be chosen, to obtain very precise results. To find a range in a pre-screen of unknown samples, a broader dilution series (1:10) is recommended. The decimal geometric series, first described by Hackenberg and Bartling 1 for use in toxicological and pharmacological studies has the advantage that independent experiments with wide or narrow dose factors can be easily compared because they share identical concentrations. Furthermore, under certain circumstances, experiments can even be merged together: A log-interval is divided into equidistant steps by a specified dilution factor (Table 1). The dosing factor of 3.16 (= 2 10) divides a log into 2 equidistant steps, a factor of 2.15 (= 3 10) divides a decade into 3 steps. The factor of 1.47 (= 6 10) divides a log into 6 equidistant steps, and the factor of 1.21 (= 12 10) divides the log into 12 steps. The production of a decimal geometric dilution series is quite simple. An example is given for the factor 1.47: Dilute 1 volume of the highest concentration by adding 0.47 volumes of sample diluent. After equilibration dilute 1 volume of this solution by adding 0.47 volumes of sample diluent and continue like this. For more information, see NIH publication No: ; Table 1: Decimal geometric dilution series divide log-intervals into comparable equidistant steps 2 Dilutions within a log interval factor ( 2 10) ( 3 10) ( 6 10) ( 12 10) Other Considerations Colored or fluorescent samples, especially natural compounds, must be tested to ascertain if they interfere with the chemistry or detection of the assay. If interference is found, an alternate detection reagent must be used. If solvents with a known toxic potential (e.g. DMSO) are used, a control with solvent alone should be included in the experimental design. If a significant effect is observed or expected, a complete dilution series of the solvent should be performed to discover the highest nontoxic concentration of the solvent in which valid results of the assay can be obtained. Assay Protocols Cell Treatment All compounds of the InstaCell Proliferation Assays are sterile and it is recommended that all work be performed under sterile cell culture conditions. However, if sterile conditions are not possible or convenient, all handling should be performed with special care. Cell culture medium and diluent buffer contain penicillin and streptomycin to suppress contamination 1. Prepare stock-solutions of your test samples at a convenient concentration. 3

4 Hydrophobic compounds can be dissolved in DMSO. However, the final concentrations of DMSO in the assay should not exceed 1%. 2. Pre-warm culture medium in a water bath to 37 C. 3. Take an InstaCell Proliferation Assay plate out of the -80 C freezer, remove the plastic foil and place it directly into a 37 C incubator. Do not allow the cells to thaw at room temperature. Keep the plate on dry ice during transport if necessary! 4. Thaw the InstaCell Proliferation Assay plate for 30 minutes. Avoid stacking and moving plates during thawing! 5. Add 75 µl of pre-warmed culture medium to each well after complete thawing of the cells. Move the plate on the surface of the work-bench in a figure 8 to ensure equal dispersion of the cells. 6. Incubate the plate in a humidified atmosphere at 37 C for at least 4 hours to revitalize the cells. Prolong the revitalization overnight if a short incubation period of the test compounds (48 hours) is planned. 7. Prepare a series of different dilutions from the stock solution of the test compounds using the dilution buffer. If the stock solution of the test substance is prepared with solvents other than water (DMSO, methanol, etc.) prepare proper solvent controls. Pre-warm diluted samples and positive control to 37 C. The samples will be again diluted in the assays plates to 50%. So prepare a double concentrated start concentration for the dilution series to reach the correct final concentration in the assay! 8 Add 125 µl of diluted samples and controls to each well of the assays plate according to the planned assay configuration. Add 125 µl of dilution buffer to wells that are not used in the assay to protect adjacent wells from evaporation. 9 Incubate the assay plate at 37 C in a humidified atmosphere. Proliferation assays are usually complete in 72 hours, but an extension to 96 hours is possible before the cells become confluent and growth inhibited. XTT Assay 1. Pre-warm the XTT solution to 37 C before staining. 2. Add 50 µl of XTT solution to each well of the assay plate. Incubate for at least 4 hours at 37 C, until a color shift from yellow to orange can be observed in untreated wells. Sub-optimal culture conditions can slow down the staining procedure! The incubation can be prolonged up to 24 hours to obtain a clear differentiation of positive (yellow) and negative (orange) controls. 3. Measure absorbance in microplate reader at 450 nm and a reference measurement at >650 nm. 4 Plot the data as percentage of the negative or solvent control value (100%). See figure 1. Trouble Shooting 1. Weak signal. Small difference between positive and negative controls Seeding density of cells is optimized for reading of the assay 80 hours after thawing the cells, assuming optimal 4

5 culture conditions. If culture conditions were suboptimal (e.g. no CO2 incubator), growth and metabolic activity might be decreased. Prolong the incubation with the staining solution (XTT) until a significant difference between positive (yellow) and negative (orange) control can be observed. Prolong reactivation period up to 24 hours before adding test samples Viability of the cells might be decreased. Control storage conditions. Assay plates have to be kept strictly at -80 C before use. Control the viability of the cells after revitalization by microscopic evaluation (attachment of the cells). 2. Large deviation of signal. If individual wells show a very high, unexpected metabolic activity, the cause is probably bacterial contamination. Control the respective wells microscopically. Do not include the data from these wells in the IC 50 calculations. Work under sterile conditions. Some colored or auto-fluorescent test compounds might interfere with the assay chemistry. Perform respective assay interference controls or use an alternative detection method. 3. Unexpected deviation of signal in wells at the edge of the assay area. Increased evaporation of medium in the border wells of the assay plate can result in discrepancies. Exclude the border wells from the assay if necessary. Add medium to all unused wells to minimize evaporation. Control the humidity in your incubator or use a closed humidified box. 4. Negative control wells and wells with the lowest concentration of test compound became pale and give no fluorescence signal Technical Specifications Assay Plate: 96-well-plate; TC-grade; F-form Cell Line: HCT-116 Human colorectal carcinoma Matrix: Cryomedium containing 10% FCS, DMSO-free Culture Medium: RPMI 1640 w/o phenol red; 45 mm HEPES; Pen/Strep (150 U / ml) Dilution Buffer: RPMI 1640 w/o phenol red; 30 mm HEPES; Pen/Strep (100 U / ml) XTT Solution: 2,3-bis(2-methoxy-4-nitro-5- sulfophenyl)-5-[(phenylamino) carbonyl]-2htertrazolium hydroxide (1 mg/ml); PMS(1%) in PBS Positive Control: 5-Fluorouracil in DMSO (5 mm) Incubation: 37 C ± 2 C, 5% CO2 and 95% humidity Test Duration: hours XTT Determination: Absorbance 450 nm / Reference >650 nm Shelf life: 6 month from date of production The plates thaw from the edge of the plates to the middle. If the plates are moved during the thawing process or are allowed to thaw before they are placed into the incubator, the viability of the cells might become reduced in the center of the plate. Carefully follow the instruction for thawing the assay plate. Don t add medium before the cells are completely thawed. Control the viability of the cells after revitalization by microscopic evaluation (attachment of cells). 5

6 USE OF PRODUCT This product contains research chemicals. As such, they should be used and handled only by or under the supervision of technically qualified individuals. This product is not intended for diagnostic or human use. WARRANTY BIOMOL International, LP makes no warranty of any kind, expressed or implied, which extends beyond the description of the product in this brochure, except that the material will meet our specifications at the time of delivery. BIOMOL International, LP makes no guarantee of results and assumes no liability for injuries, damages or penalties resulting from product use, since the conditions of handling and use are beyond our control. BIOMOL International, LP 5120 Butler Pike Plymouth Meeting, PA Phone: (800) (610) Fax: (610) URL: References: 1. U. Hackenberg and H. Bartling. Arch. Exp. Pathol. Pharmakol NIH publication No: ; 2001). 6