Dear Muhammad Irsyad Ramadhan

I briefly wanted to reach out concerning your inquiries. As you did not lay out all details for your experiment, I had to make some assumptions.

As you did not specify your MACS kit, if not already please consider

https://www.miltenyibiotec.com/CA-en/products/indirect-cd133-microbead-kit-human.html

I have used their kits with great success on hematopoietic stem cells or immune cells and simply followed manufacturer's instructions.

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Furthermore, if you have access to different cell lines, please consider OVCAR-3 cells as these seem to have a more stem cell like phenotype (when assessed by CD133 expression). For a comparison please see, Figure 1. from this attached paper. I actually think that your low yield of cells, is due to the fact that SCOV3 cells predominantly do not express CD133.

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In addition, the TGFb pathway is overactive in CD133+ (ovarian) cancer stem cells, so simple things like TGFb treatment may also increase your yield even in cell lines that stain very little for this marker.

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However, I understand that this may interfere with your experimental design.

I hope that helps.

All the best & good luck with your experiment,

Michael

Dear Muhammad Irsyad Ramadhan,

I have several questions for you.

1. Do you measure the amount of CD133+ cells in your sample, before you start selection? That is necessary so that you can calculate your yield.

2. Do you follow the instructions by the company, as they are described in their kit insert? For example, when they say ice cold solution, you need to have it exactly like that.

I have used their CD34+ cell selection beads about 25 years ago, I had low recoveries like 35% or so, then I have been informed that wash buffer had to be icy cold and I had to push the plunge strongly to have better results. After applying these simple solutions, my recoveries went up to >95%.

My first suggestion to you will be to contact the technical support of the vendor, discuss the difficulties with them and go on with your experiments. They were very helpful 25 years ago, I expect them to even better now.

With best wishes,

Hi Michael Förster, thank you so much for your reply, and yes our lab has been using the same kit. Furthermore, I do think, your point regarding the CD133 differential expression across cell lines seems legit! So, again, thank you for sharing the knowledge.

Also, thank you Gulderen Yanikkaya Demirel for the technical suggestions, but yeah I already done all the instructions, so an intrinsic culture factor is more likely to be the cause. But, I'm really intrigued with your question #1, because I don't know how can I count it exactly.

Dear Mohammad,

You can count by automated cell counters used for cell culture samples. A hand held one is Scepter. If you think they are expensive I think the manual hemacounters like Neubauer chamber are still around, you may use one of them.