Hi,

yo can do several things

1. Treat your cells and analyse cell cycle/ apoptosis by FACS

2. Treat your cells and analyse by viability assay (e.g. viability assays from promega)

both methods have pro's and con's bu for screening I would prefer viability assays because you can quntifiy the strenght of the effect more precisely

you can use MCF 7 and MDA-MB 231 cell line

to test the cytotoxicity, its best to first go with viability assay like MTT, LDH, cystal violet assay. 

I suggest to think about a) method, b) general vs specific toxicity, and c) type of toxicity of your molecules/drugs.

Methods to use: Suggested by Paromita and Sven methods are good. I would go first  with MTT, as it is simple and easy to use for screening.

General vs specific toxicity: One may use a single cell line, e.g. MCF7 or MDA-MB-231, but it would be only about general toxicity with specificity related only to the used cell line. This would tell you whether the drug/molecul kills cells in a therapeutic range, e.g. below of 10 microM concentration. If you plan to use the molecule/drug for a clinic, you would have to do a cell panel. The panel has to have normal breast epithelial cells (e.g.HMEC or primary cells), immortalized (e.g. 184A, MCF10A), conditionally tumorigenic (e.g. MCF7), metastatic (e.g. MDA-MB-231), fibroblasts, hepatocytes, keratinocytes, endothelial cells. You may include also cells of other types of cancers. You would have to do titration of the drug/molecule concentration and check different times of treatment for cytostatic and cytotoxic effects. MTT usually gives a good read-out..

With obtained curve of cell growth upon treatment with different doses and times, it is easy to calculate an effect, e.g. EffectiveDose50, ED80 and ED100. Shape of the growth curve will also tell you whether you have cytostatic or cytotoxic effects.

Type of toxicity: This is usually done after establishing dose/time profile of the molecule/drug. You would check which mechanisms are engaged by the drug/molecule.

Hope my comments are useful.

Good luck,

Serhiy Souchelnytskyi

www.serhiysouchelnytskyi.expert

Thank you this is great...

With Regards to the cell lines:

Can we take primary cancer  tissue from a tumor and test the viability without using the cell lines. 

I am doing several cases of breast cancer surgery, here in Sri Lanka.

Dear Kithsiri,

That would be possible, but there are many caveat that you need to pay attention. For example the need for informed consent and the secrecy of the patient identity (which is an ethical issue and the most crucial one), second is the problem of subculturing the cancer cell into stable cell line, third is you have to know exactly what kind of cancer is this, is it Luminal A, Luminal B, HER2+, or TNBC? It would be great if you even have the gene expression microarray result.

But before you do, I think it would be highly advised to do preliminary cytotoxicity assay on commercially available breast cancer cell lines.

Hope that help,

Radif

thanks, Radif, 

I am new to this kind of research. appreciate your help.

 @ Serhiy, you have suggested that the efficacy of killing cells by the drug should be in therapeutic range. May i know what is the therapeutic range of a drug under invitro and in vivo conditions?

I guess wht Sehiy wants t tell is that at least fo rknown compounds the used concentrations should be in range of known IC50 values. It makes no sense to oferexeceed this value lets sa 10 times and therefor think it is an compound specific effect. Further more you have to closely monitor potential "side effects" cause by the diluent (e.g. DMSO or methanol are dose dependent toxic) and for this use controls with the amount of diluent or ideally a non active version of you compound.

Regards

Sven

Talk to Professor Jayantha Rajapakse of our Vet Faculty.

Ill send my proposal to you sir. This is on a nanopaticle...

Dear Kithsiri Senanayake 

I have used 4T1 cell line (mice breast cancer cells) and applied MTT assay to evaluate cytotoxicity of my agents. below is the procedure:

Cells were cultured in RPMI 1640 medium supplemented with 10% (v/v) FBS and 100μg and 100 IU of  antibiotics (Pen-Strep) and then incubated at 37 ºC, 5% CO2, and95% humidity for 24 h. Cellular growth in the presence or absence of drug was evaluated by measuring the degree of mitochondrial function and reduction of the tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to formazan crystals (Carmichael et al. 1987). Cells were adjusted to 1x106 cells per ml and seeded into a 96-well plate with 100μl in each well and incubated for 24 h. Thereafter, drug were applied to the culture well to reach the final concentrations of 0–100 µg/ml. After 24 h of incubation, the cells were treated with 20 µl of the MTT reagent at a concentration of 5 mg ml-1 in PBS and incubated for a further 4 h. Consequently, the media was discarded and 150 µl DMSO was added to solubilize the formazan crystals. After 5 minutes shaking to mix the formazan in the solvent, the optical densities were determined at 550 nm. MTT assay was performed in triplicate.

Good Luck

Its Great, Dr Yazadi...

A colorimetric assay cannot assess "cytotoxicity".

Cytotoxicity means "cell killing effects".

A colorimetric assay can only bring "growth inhibition information" because it is a relative test in which you compare the ODs of a treated condition to the ODs of a control condition arbitrarily scaled at 100%.

When you obtain a concentration (for a given compound) decreasing by 50% the global growth (after x days), you do not know whether you killed 50% of the cells (cytotoxic effects), whether you inhibit 50% of the cell proliferation (cytostatic effects), whether you detach 50% of the cells (anti-adhesive, i.e. "in vitro antimetastatic" effects), etc..., etc...

Once you have determined the IC50 concentration for a given compound on a given cell line, you must use complementary biochemical and/or morphological techniques to determine whether your compound is cytotoxic, cytostatic, anti-adhesive, etc..., etc...

Some of the attached articles could also be of help.

For example, in the Mathieu et al. (2009 and 2015) articles, the MTT test-related GI50 concentrations relate to actual cytotoxic effects.

In the Lefranc – Nunzo et al. (2013) article, the MTT test-related GI50 concentrations relate to cytotoxic effects that in turn do not relate to apoptosis … This means that each cytotoxic effect does not “universally” translate into pro-apoptotic ones (see the Kornienko et al. (2013) article).

In the Van Goietsenoven et al. (2010) article, the MTT test-related GI50 concentrations relate to cytostatic effects, neither to cytotoxic nor to pro-apoptotic ones.

Be aware that you cannot always translate the MTT test-related growth inhibition of a given compound into a precise GI50 value. Some compounds reach a “plateau” of inhibition (see Lefranc – Nunzo et al., 2013).

Lastly, you can also have "false" data generated with the MTT colorimetric assay (see the Chan et al. (2013) attached article).

The attached articles by Galluzzi and colleagues (2009, 2012, 2015) perfectly illustrate how mixing various tests to be able to actually “speak” about cytotoxicity.

Best regards

Robert

Thanks Dr. Robert kiss for detail clarification.