Hi, I use to prepare a stock 1mM in ETOH and store -20. Then, I use 250nM final in culture. I thin that timing is model-dependent. I change media everyday for 3 days in a row in my model and this gave me the maximum effect (p53 restoration in nearly 100% of cells). in your system should be different, the best way to check gene reduction in time course experiments, then optimize the time of treatment.

good luck!

Thank you for your response Michela.

For anyone still wondering how to use 4-OHT: I have used 4-OHT in vitro to successfully delete my gene of interest in bone marrow-derived macrophages (BMDMs). The protocol (attached) worked reasonably well (>4 fold reduction) in gene expression. The protocol is written in short hand (assuming that one is familiar with BMDMs and their generation). If there are any questions please let me know.

Best of luck!

Dear Liberty

thank you for sharing this informations. I have a arc-gfp mice that can be induced upon tamoxifen injection. I am trying to establish neuronal primary cell culture and to induce the arc-gfp expression upon 4OH treatment. I would like to know if you have some hints for me. I would like to use 40h at final concentration 1µM in culture to induce the arc-gfp. Do you know for How long do I leave the 4-OHT in the medium to have get a signal?

Thank you.

Francesco

Hi Francesco,

Apologies but I am not knowledgeable in your cells therefore I can't provide many hints/tips other than what is in my previous answer. Others have induced gene deletion with a concentration of 1uM 4-OHT but again you would have to examine this in your specific cells. In my protocol I maintained my cells in medium containing 2 uM 4-OHT for seven days but like I said, I am not knowledgeable in your cells therefore this time point may or may not be suitable. I recommend you perform pilot studies to examine efficacy of gfp expression post 4-OHT treatment and examine toxicity of 4-OHT to your cells.

I'm sorry that I can't be of more help.

Thank you anyway. I will find a solution just playing around.

Best,

Francesco

Hi Liberty,

Thanks for your information. I am planning to do a similar experiment on BMDMs. I do however have few questions:

1. How much BM cells did you seed in 6 well/12 well for BMDM culture?

2. Did you evaluate the amount of macrophages after differentiation (7th day) by surface markers expression (Flow cytometry). I cultured BMDMs and got 85-87% of macrophages on 6th day (I read it should be around 95-98%).

3. I am planning to stimulate my tamoxifen treated BM-derived-macrophages by LPS, CpG, Pam3CSK, so when do you think is the best time time to stimulate the BMDMs. Is it after differentiation (6-7th day)?

Hi Bhakti,

1. I seed 2 million BMDMs per well for 6 well plates on the 7th day and let them adhere overnight. The following day (8th) I remove the medium with LCM and perform my experiments in medium without LCM.

2. Yes I have. On the 7th day I seed cells as above onto coverslips then perform IHC for CD68 and F4/80 and perform counts of positively stained cells (usually >99%). I have not tried FC.

3. This is an interesting question. In my time working with BMDMs, I have found them to be unstable when LCM is removed from the medium. Therefore I perfomed a few experiments to examine when BMDMs are most mature and stable and I found that to be on the seventh/eighth day of culture. Therefore, I usually seed BMDMs onto six well plates on the seventh day and perform my experiments in LCM-free media on the 8th day.

These answers are not exhaustive. If you would like the rationale behind the answers, please message me with your email and I will answer at length.

Thank you.

Thanks alot Liberty. I have got success in my first two question-related experiments. However, I have few more questions. I shall get back to you by email you.

Thanks again.

Hi Liberty ,

Thank you for your information. I'm doing a similar experiment on BMDM. But I have some questions,

1, no matter what concentration I use, my BMDM don't die. ever I use 2uM, 3uM，4uM，5uM

2，I want to know the purpose that you use 4-OHT with heat. Because you wan to melt quicklly?

3, I find low density cells will delete better the target gene, but I look your cell density, could you tell me your rate of KO? my rate of KO is only 50%

Hi Juan,

1. May I ask, do you maintain mature BMDMs in 4OHT or the progenitor cells as per my protocol? In my experiments, BMDMs did not die with 4OHT treatment if I started the treatment at 7 days (mature macrophages). I maintained mature BMDMs in 2, 4, 8 and 16 uM for several days and like you say there was little death as assessed by propidium iodide uptake. However, when I maintained bone marrow progenitor cells in 2 uM 4OHT, the amount of mature BMDMs that had formed by day 7 was significantly less than those maintained in vehicle.

2. According to the datasheet (https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Datasheet/7/h7904dat.pdf) , 4OHT powder will dissolve in ethanol (20mg/mL) with heating producing a clear faint yellow solution. Please check my protocol (can be found in one of my earlier answers) for more details of how to make up 4OHT

3. In my experiments, I use 4OHT to induce gene depletion as the cells mature into BMDMs. My depletion efficiency is ~4 - 8 fold as assessed by mRNA expression of the target gene.

I hope this answers your questions but if not please don't hesitate to message me here or email me.

Edit: my email is [email protected]

Best wishes

Hi Liberty ,

Thank you so much. tomorrow I will do this experiment according to your protocol. I wish I can get the same result .

Hi Liberty

Thank you for sharing your protocol. However, I still have a question of dissolving after you answered others' question. I also use the 4-OHT(h7904) of sigma and the 4-OHT is in a smaller tube in the bottle.

1.After I pipette the 100% EtOH into the tube , it seems some 4-OHT plugged in my tip or attach the wall. More I tried to pipette it down, more stick on the wall. Do you see this problem when you dissolving?

2.I still heat to dissolve 4-OHT, but I didn't see the color change after dissolving. Is the reason that losing too much 4-OHT during pipetteing?

Hi Wei yu Lai,

No problem, it has been awhile since I last did this, but I do not remember any issue with dissolving the powder. It did take a lot of time, heat and continuous agitation for the powder to dissolve. I suggest you try centrifuge the power to collect it all at the bottom of the container before you attempt dissolving?

As for the color change, are you referring to what sigma say should be a yellowish colour of the stock solution? If so, depending on the volume you dilute in you may see less of a yellowsih colour. I don't remember the solution being very yellow but that being said sigma mention that it is a very faint yellow. Also, I dissolved the powder in the brown glass container it came in so it would have been hard to tell its colour. After dissolving the powder I then pipetted a small amount of the stock solution into a larger volume of PBS as per my protocol so it may have been difficult to notice the faint yellowish colour that the sigma protocol mentions.

I hope that helps.

Hi Liberty

Thanks for your suggestion! I'll observe the color carefully after dissolving next time. Hope it would go well next time.

And here is another question during diluting with PBS. Do you see 4-OHT precipitate? I found some people also have this problem when they diluted, and I wonder if the solution is also heating 4-OHT/PBS until it dissolve?

The link is another discussion I found.

https://www.researchgate.net/post/How_can_I_avoid_4-OHT_stock_concentration_of_10mM_in_DMSO_from_precipitating_when_added_to_zebrafish_E3_media_for_a_final_concentration_of_10uM

My question on the topic is how often do you add 4(OH)TAM in the cell culture? I usually add 1μM of 4(OH)TAM daily.

Hi Wei yu Lai,

Apologies for the delayed response. I dissolved the powder in molecular biology grade ethanol, not PBS. Then, I swirl the container in a water bath set to 37 degrees celsius until dissolved if there is some precipitate. I further dilute this stock in PBS.

Hope this helps.

Hi Ioannis Kokkinopoulos,

When working with BMDMs I added 4OHT twice - once on first day of cell culture and replenished on the third day. Take a look at one of my previous answers for a more complete protocol.

If your cells can tolerate what you're suggesting, fine. Be sure to check toxicity etc.

Hope this helps.

Hi Liberty Mthunzi, thank you for your answer. So, I guess you add 4(OH)TAM every 48 or 72 hours? Have you tested if there is a difference between daily and more intermittent treatments?

My BMDM cell culture protocol is seven days so I add the 4OHT only twice.

I haven’t tested that because prior literature suggested to me that daily treatment of 2uM 4OHT would be toxic to BM derived cells.

Liberty Mthunzi, I followed your protocol and my 4-OHT came out of solution when I diluted the 51.6mM to 2.5mM in 1X PBS (solution became milky white). Does this happen to you?

Most other protocols dilute in EtOH or DMSO, do they not?

Hi Matthew Ingalls,

To be honest, it is has been awhile since I did this but I don't remember this happening. Potentially, you could use EtOH as the diluent for further dilutions (after 51.6mm) and see if it helps. Alternatively, you could try use heat and agitation. From memory the powder doesn't dissolve easily (even in EtOH) so heat and agitation was necessary.

Sorry I can't be of more help. Let me know how you get on please.

What is the optimum concentration of 4-OHT? I added 20 uM 4-OHT in Chinese hamster cell line. I checked the cell by fluorescence microscopy. Most of the cells has been died!

20uM of 4(OH)Tam is too high for in vitro administration. Depending on the cell line used, 0.5 uM to 2uM will be the optimal, per 24 hours.

This is because excessive 4(OH)Tam will induce growth arrest and potential apoptosis.

@ Ioannis Kokkinopoulos Thank you for your suggestion. I will try to conduct new experiments with lower concentration as you recommended.

I would like to know the optimum concentration of 4-OHT in cell growth. Do I need to apply different concentration of 4-OHT in the same day of re seeding the cell? Or the next day when the cell is more confluent?

Hi Md Rashidur Rahman,

Sorry for the delayed reponse. You will need to titrate 4-OHT to figure out the optimum dose for your specific cell culture conditions (cell type, length of culture, cell culture medium etc). In my experiments with BMDM cell culture what worked best was 2 uM concentration of 4-OHT for seven days and an exchange of medium once on day three of culture as stated in the protocol I previously provided.

Best,

Liberty