In agreement with all the previous recommendations, induction of apoptosis varies depending of the cell line and the toxicant utilized. In many cases, you need to find out the adequate conditions according with the method and time that you are going to use to measure apoptosis. For instance, to induce externalization of phosphatidylserine (PS) after ~24h (from 18 to 24h) we routinely use a range of H2O2 from 100 µM to 2 mM to be monitored by using annexin V-FITC. However, to detect PS translocation after 48h to 96h 1 mg/ml G418 is useful. If you are going to detect caspase-3 activation and PARP-1 cleavage, after 6 and 8 h, 2 μg/ml camptothecin is recommended. Additionally, if you are going to detect early facets of apoptosis (4h), like mitochondrial depolarization, 50 μM of cyanide m-chlorophenylhydrazone (CCCP) is suggested. Also, to induce caspase-8 activation after 4 h of incubation, 2 mM of H2O2 could be useful.

For more details check Robles-Escajeda et al. (2013). PLoS ONE 8(9): e73508. Good luck!!!

Hi Qinglin:

I have never worked with that cell line, but I get nice apoptotis induction of my cells with DMSO (5% final concentration for 5x10(4) Raji cells/200 ul, in 48 h cultures). I hope that this suggestion works in your case.

Good luck!

I have no esperience with this cell line. A convenient protocol to induce apoptosis in some cell lines is incubate the cells at 42ºC for 30 minutes. This portocol does not require the removal of the apoptotic stimuluas in further incubations. Hope it works in your cell line!

You can try a treatment with different dilutions of Sodium Butyrate or Staurosporine. Both compunds are apoptosis inductors.

I have not tried to induce apoptosis in this particular cell line but usually staurosporine is quite effective on almost every cell line.

How are you measuring apoptosis? In some cases the main problem is not the induction protocol, bit the apoptosis assay.

The topoisomerase inhibitor etoposide (also known as VP16) is a very effective apoptosis inducer in many cell types

I did never use this cell line. Usually, stravation of cells (simply culturing cells without serum or with low serum concentration), gamma-irrradiation, phorbolesters (PMA) at high doses (and generral several other drugs, see the replies to your query in this page), anti-Fas antibodies of IgM isotype or other anti-death receptors antibodies can induce apoptosis. To use antibodies you should test cells for the expression of the receptors and then if the receptors is expressed you should (usually) cross-link the receptor (using antibodies of IgM isotype the cross-linking is optimal without any other kind of cross-linker as goat anti-mouse antiserum). Please try different doses and time of exposure to an apoptotic stimulus, of course, to get good results. Protocols to induce apoptosis are well described in several works that you can easily download from the web.

For years we are using Celastrol (1-10µM final) and it worked on all the human cell lines we tested (Moreira-Tabaka H et al. PLoS One 2012;7:e34184) so far.

Hi Qinglin

a search in PubMed about 4T1 cell line + apoptosis might perhaps give you some ideas. Treatment with known antitumor drugs (etoposide, arsenic trioxide, doxorubicin, cisplatin) may be good solution because the paradoxic behaviour of tumor cells. Naturally, cancer cells are resistant to death but highly susceptible to antitumor drugs!!! so this is the reason why this kind of drugs are use in the clinic.

Good luck

Adrian

Although with different type of cells as yours, we have analyzed death´s cells by apoptosis with different apoptotic stimuli like 1–10 μg/ml of commercial C-Reactive Protein for 4–12 h and 10 μmol/l H2O2 for 12 h or 100 μmol/l 7β-hydroxycholesterol for 48 h

TKI258, an FGFR tyrosine kinase inhibitor, has been reported to induce 4T1 apoptotic death via blockade of the phosphoinositide 3-kinase/AKT pathway.

Dey et al. (2010) Cancer Res. 70: 4151-4162

Good luck!

What is the aim of your study?

Grape seed proanthocyanidins induce apoptosis and inhibit metastasis of

highly metastatic breast carcinoma cells.

Sudheer K.Mantena, Manjeshwar S.Baliga and

Santosh K.Katiyar

Carcinogenesis vol.27 no.8 pp.1682–1691, 2006

doi:10.1093/carcin/bgl030

Advance Access publication April 5, 2006

In agreement with all the previous recommendations, induction of apoptosis varies depending of the cell line and the toxicant utilized. In many cases, you need to find out the adequate conditions according with the method and time that you are going to use to measure apoptosis. For instance, to induce externalization of phosphatidylserine (PS) after ~24h (from 18 to 24h) we routinely use a range of H2O2 from 100 µM to 2 mM to be monitored by using annexin V-FITC. However, to detect PS translocation after 48h to 96h 1 mg/ml G418 is useful. If you are going to detect caspase-3 activation and PARP-1 cleavage, after 6 and 8 h, 2 μg/ml camptothecin is recommended. Additionally, if you are going to detect early facets of apoptosis (4h), like mitochondrial depolarization, 50 μM of cyanide m-chlorophenylhydrazone (CCCP) is suggested. Also, to induce caspase-8 activation after 4 h of incubation, 2 mM of H2O2 could be useful.

For more details check Robles-Escajeda et al. (2013). PLoS ONE 8(9): e73508. Good luck!!!

Yes, I agree with many of the comments above. I would use the topoisomerase inhibitor, camptothecin. A ref with 4T1 cells and this agent will be useful to you:

Response of Mouse Breast Cancer Cells to Anastrozole, Tamoxifen, and the Combination

Xanthopoulos JM, Romano AE, Majumdar SK - J. Biomed. Biotechnol. (2005)

Hi, could you read our last paper Urueña 2013. BMC complementary and alternative medicine, and if you have specific questions please do not hesitate.

I have been using 4T1 cells for many years. And if you look at our publication, you can see that we observed DNA fragmentation in response to proteasome inhibitor bortezomib or MG132 in 4T1 cells. Also, you should know that 4T1 is a p53-mutant cell line (Yerlikaya, A. et al., Tumor Biology, 2012). And if your agent induces apoptosis in a p53-dependent manner, then it may be better to include a positive control for your apoptosis induction.