Ic50 determination protocol

Cell Viability is a common technique used by biochemists who are studying oncology and pharmaceutics. The most common use for cell viability studies is when determining the IC50 for a cytotoxic compound in cell culture. However, as you can expect, there are a lot of different times when you need to know if your cells are alive.

In this method guide, we will walk through the theory behind all these methods and then end with a protocol for the MTT assay. Try it out here …. In general, to measure cell viability, you need to incubate cells with a reagent and measure the conversion of your reagent into a product. If lots of cells are alive, most of your reagent will be converted. If lots of cells are dead, then your reagent will only be partially converted. For the MTT assay, the reagent used is 3 - 45 -dimethylthiazol- 2 -yl - 25 -diphenyltetrazolium bromide tetrazolium.

Because of its positive charge, MTT can enter viable cells and non-viable cells with ease. Upon conversion, the Formazan product precipitates inside cells near the cell surface and can be detected using a spectrophotometer.

Note: MTT only needs an intact and functioning mitochondria to be converted so it is a metabolic assay and not a proliferation assay. The assay technique is very simple:. Take a look below to understand these steps:. So, if you add a cytotoxic material which reduces mitochondrial efficiency, you might get weird results. The other major cell viability assays that are used in research include:. Cell Titer Blue : Similar to the MTT Assay, this assay involves incubating cells with resazurin blue and forming resorfurin pink after the cells metabolize it.

This is also a great high throughput assay!

EC50 and IC50 Determination in Excel

Because trypan blue is a charged dye, it cannot permeate through living cells. So, simply incubating cells with trypan blue and looking under a microscope allows you to visually determine the of viable cells unlabeledof non-viable cells blueand the of damaged cells slightly blue.

Viability is just the ratio of live cells divided by total number of cells. The disadvantage with this method is that all you test is the membrane integrity of the cells. The general principle however is all the same.

Here are some images describing the above methods:. I am a PhD Bioengineer specialized in utilizing heparan sulfate and heparin for drug delivery to brain tumors. My expertise lies in the interface between polymer chemistry, protein biochemistry, and cellular biology.

View all posts by Karthik Raman, PhD.Vorozole and letrozole are third-generation aromatase cytochrome P 19A1 inhibitors. Pretreatment with letrozole does not affect the binding of vorozole in the liver.

Since CYP3A4 makes up the majority of the CYP content found in the human liver, and vorozole inhibits it moderately well but letrozole does not, CYP3A4 is a good candidate for the protein that [ 11 C]-vorozole is binding to in the liver. Vorozole and letrozole Figure 1 are nonsteroidal, triazole-containing compounds that are competitive, reversible, third-generation aromatase CYP19A1 inhibitors [ 12 ]. Both vorozole and letrozole were initially developed and underwent clinical trials as antineoplastic agents [ 3 ].

Letrozole Femara is currently used in the treatment of breast cancer [ 4 ] but vorozole was not pushed forward after phase III clinical trials because it did not show any significant advantage over the current drugs [ 5 — 7 ].

Methodology

However, vorozole, labeled with 11 C, is currently being used as a tracer for positron emission topography PET imaging to study CYP19A1 distribution in living animals [ 8 — 13 ]. In vivo studies have shown that [ 11 C]-vorozole binds regionally specifically to CYP19A1 in rhesus monkey [ 16 ], baboon [ 1117 ], and human [ 8 ] brain. The brain accumulation has been shown to be specific by being blocked by both vorozole and letrozole [ 81617 ].

However, when [ 11 C]-vorozole is administered to rats [ 10 ], rhesus monkeys [ 14 ], baboons [ 17 ], or humans [ 18 ] by IV, some of it binds to the liver. It has been shown that this binding in the baboon and human liver is not caused by CYP19A1 because pretreatment with letrozole does not block its binding [ 1819 ].

While vorozole has been shown to be selective against other cytochrome Ps- CYP- dependent reactions in steroid biosynthesis [ 6 ], there is limited data available on other CYPs, especially those found in the liver.

Many imidazole and triazole ring-containing inhibitors of CYPs form a noncovalent ligand interaction with the ferric ion heme and therefore have the potential to inhibit multiple isoforms [ 21 ].

Since vorozole has been shown to bind to the liver and pretreatment with letrozole does not block this binding, by determining and comparing the binding affinity of both vorozole and letrozole on a series of liver CYPs, we can potentially identify the protein that is responsible for vorozole binding in the liver. This CYP can be identified by having a high binding affinity to vorozole but not letrozole.

These assays use nonnatural coumarin substrates that are converted into fluorescent products by the CYPs. Louis, MO. Vorozole and letrozole were provided by Brookhaven National Laboratory. The data were fit to sigmoidal dose-response curves with nonlinear regression and IC 50 values calculated using GraphPad Prism 5. IC 50 values were converted to values using the Cheng-Prusoff equation and literature values when available. Since vorozole contains a triazole ring and binds to the heme of CYP19A1, it is likely that vorozole can bind to the heme of other CYPs.

Therefore we can use letrozole as our negative control when searching for the protein responsible for vorozole accumulation in the liver. To show that our assays are valid, we confirmed that vorozole and letrozole are very potent inhibitors of CYP19A1. It was found that vorozole and letrozole bind equally poor to CYP1A2 1. While vorozole is slightly more potent than letrozole on CYP2A6 4. However, there is conflicting evidence as to whether CYP1A1 is expressed in the human liver [ 31 ].

Cytotoxicity & Cell Viability with MTT Assay Protocol

Even if CYP1A1 is expressed, it is in such small quantities [ 32 ] that it is an unlikely candidate. Therefore, there is at least a fold difference in the potency of vorozole and letrozole on CYP3A4. This data, combined with the fact that CYP3A4 makes up the majority of the CYP content found in the liver [ 20 ], implies that CYP3A4 is a good candidate for the enzyme that [ 11 C]-vorozole is binding to in the liver but letrozole does not block. The inhibition potential of vorozole and letrozole on some other liver CYPs has previously been reported in the literature.

Since there is at least a 5-fold difference in potency between vorozole and letrozole and vorozole inhibits CYP1B1 moderately well, CYP1B1 should not be overlooked. With this information, our collaborators at Brookhaven National Laboratories used a CYP3A4 inhibitor ketoconazole to see if it would block the binding of [ 11 C]-vorozole to the liver.

When [ 11 C]-vorozole was given with a pretreatment of ketoconazole, the liver pharmacokinetics of [ 11 C]-vorozole binding was altered [ 19 ]. Therefore CYP3A4 is a likely candidate for the protein responsible for binding vorozole in the liver.

Pretreatment with ketoconazole, a CYP3A4 inhibitor, affects liver pharmacokinetics of [ 11 C]-vorozole binding, further confirming that CYP3A4 may be responsible for [ 11 C]-vorozole accumulation in the liver. The authors declare that there is no conflict of interests regarding the publication of this paper. Vorozole and letrozole were kindly supplied by Dr.ISO specifies quantitative test methods to determine the antibacterial activity of all antibacterial textile products including nonwovens.

ISO is applicable to all textile products, including cloth, wadding, thread and material for clothing, bedclothes, home furnishings and miscellaneous goods, regardless of the type of antibacterial agent used organic, inorganic, natural or man-made or the method of application built-in, after-treatment or grafting. Based on the intended application and on the environment in which the textile product is to be used and also on the surface properties of the textile properties, the user can select the most suitable of the following three inoculation methods on determination of antibacterial activity:.

Check out our FAQs. Buy this standard. Based on the intended application and on the environment in which the textile product is to be used and also on the surface properties of the textile properties, the user can select the most suitable of the following three inoculation methods on determination of antibacterial activity: a absorption method an evaluation method in which the test bacterial suspension is inoculated directly onto specimens ; b transfer method an evaluation method in which test bacteria are placed on an agar plate and transferred onto specimens ; c printing method an evaluation method in which test bacteria are placed on a filter and printed onto specimens.

Please note that paper format is currently unavailable. CHF Buy. Life cycle A standard is reviewed every 5 years 00 Preliminary. Final text received or FDIS registered for formal approval. Proof sent to secretariat or FDIS ballot initiated: 8 weeks. Close of voting. Proof returned by secretariat.

International Standard under periodical review. Got a question? Customer care. Keep up to date with ISO Sign up to our newsletter for the latest news, views and product information Subscribe. Store Standards catalogue ICS 07 English French.Drug interactions due to efflux transporters may result in one drug increasing or decreasing the systemic exposure of a second drug.

The potential for in vivo drug interactions is estimated through in vitro cell assays. Variability in in vitro parameter determination e. The objective of this study was to investigate variability in in vitro inhibition potency determination that may be due to calculation methods. In a Caco-2 cell assay, the absorptive and secretive permeability of digoxin was measured in the presence of spironolactone, itraconazole and vardenafil.

From the permeability data, the efflux ratio and net secretory flux where calculated for each inhibitor. IC 50 values were then calculated using a variety of equations and software programs. The resulting IC 50 values varied according to the parameter evaluated, whether percent inhibition or percent control was applied, and the computational IC 50 equation. This study has shown that multiple methods used to quantitate the inhibition of drug efflux in a cell assay can result in different IC 50 values.

The variability in the results in this study points to a need to standardize any transporter assay and calculation methods within a laboratory and to validate the assay with a set of known inhibitors and non-inhibitors against a clinically relevant substrate.

The online version of this article doi Recognizing the potential for drug—drug interactions DDI is an important factor in the development and regulatory review of a new drug 1 — 3. Transporter-based DDI may be due to competition for a transporter-binding site by a competitive substrate or an inhibitor or a change in level of transporter expression from an inducer.

The objective of DDI studies is to determine the potential for clinical interactions between an investigational drug and other drugs that may be co-administered. Early identification of compounds that are transporter substrates or inhibitors has become a routine task during the optimization and selection of drug candidate 5.

An integrated in vitro to in vivo approach can aid in determining the need for in vivo drug interaction studies. Variability in in vitro parameter determination, such as IC 50 or K i values, among laboratories may lead to different conclusions for in vivo interaction projections using universal criteria such as those proposed in the FDA draft and EMA drug interaction guidances 23. Therefore, predictability of in vitro assays is important, as costly clinical studies might be initiated based solely on the in vitro results 1.

Therefore, it is important to understand the sources of variability and to use standardized methods within a laboratory to minimize variability.

Bidirectional assays are the most direct and accepted models for evaluating the potential of new drugs as substrates or inhibitors of efflux transporters 25 — 7.The concept of an absolute IC50 is not standard, and many find it not to be useful. But if you do, it is not hard to fit a curve to determine it. This EPA documentgive the needed equation which I have generalized a bit, so not require that the data already be normalized.

Note the distinction between the parameter Bottom and Baseline. Bottom is the Y value of the bottom plateau of the curve itself. You'll definitely want to constrain Baseline to be a constant value based on controls. You may also want to constrain Top. Download the Prism file that fits that equation to make the graph shown above.

When fitting data to that equation, don't forget to constrain Baseline and Top to appropriate values determined by controls. These data are fit to a simpler equation where Baseline is set to equal zero, and Top is set to equal These are hard wired into the equation, so you don't have to remember to constrain those two parameters to constant values. Here is an alternative approach you can use if your data are normalized. It does not require entering a user-defined equation.

Make sure that your data are normalized to some controls. At the bottom of the data table, add a new row of data. Enter 50 into each Y column. Leave X blank for this row. Use nonlinear regression to fit the data to the log inhibitor vs. On the first fit tab of the nonlinear regression dialog, check the option: " Interpolate unknowns from standard curve. On the Constrain tab, consider constraining Top to have a constant value of The IC50 reported as part of the main results table will be the relative IC This is the absolute IC All rights reserved.

This guide is for an old version of Prism. Browse the latest version or update Prism. Fitting a dose-response curve to find the absolute IC50 The concept of an absolute IC50 is not standard, and many find it not to be useful.

An alternative approach for normalized data Here is an alternative approach you can use if your data are normalized. Scroll Prev Top Next More.Two types of neuraminidase NA inhibition assays are commonly used for determining influenza susceptibility to the NA inhibitor NAI antivirals: fluorescence-based FL and chemiluminescence-based CL assays.

Both types of assays have advantages and disadvantages see summary below.

ISO 20743:2013

Various software programs are available for the calculation of IC50 values from NA enzyme inhibition assay data. Variations in IC50 values may occur as a result of differences in the software programs.

Laboratory methodologies for testing the antiviral susceptibility of influenza viruses - HOME Meetings of the WHO working group on surveillance of influenza antiviral susceptibility.

ic50 determination protocol

Health Topics. Year of the Nurse and the Midwife About Us. Skip to main content. Laboratory methodologies for testing the antiviral susceptibility of influenza viruses: Neuraminidase inhibitor NAI Phenotyping: neuraminidase inhibition assays Two types of neuraminidase NA inhibition assays are commonly used for determining influenza susceptibility to the NA inhibitor NAI antivirals: fluorescence-based FL and chemiluminescence-based CL assays.

Summary of the available NA inhibition assays. You are here: Influenza Laboratory methodologies for testing the antiviral susceptibility of influenza viruses.Analyze, graph and present your scientific work easily with GraphPad Prism.

No coding required. Home Support. Prism can easily fit a dose response curve to determine the IC Note that the X values are logarithms of concentration. Prism offers built-in equations designed to handle X values as either concentration OR log concentration. Be sure you select the correct equation when performing nonlinear regression!

ic50 determination protocol

If you'd like to convert concentration values to log concentration values - or vice versa - you can use Prism's Transform analysis to convert the X values. Also note that this sample data set includes unknown values.

Prism can interpolate these X values. Click Analyze and then Nonlinear regression. Or click the Nonlinear Regression shortcut button just above the Analyze button.

On the Nonlinear regression dialog, open the "Dose-Response -- Inhibition" family of equations, and choose "log inhibitor vs. At the bottom of the dialog, check the option to "Interpolate unknowns from standard curve".

ic50 determination protocol

Click OK and view the results. View the graph. More information: More tutorials on curve fitting Learn about the equation used to fit to the data. Interpreting nonlinear regression results How exactly is the IC50 defined?

Relative vs. Explore the Knowledgebase.


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