According to ATCC ..."To prevent loss of myoblastic cells, cultures should be subcultured before they become confluent, and the line should be recloned periodically with selection for myoblastic cells".
Could you please let me know how to select and differentiate the myoblastic cells from others? Until now, I'm using H9c2 cells (always keep them at less than 70% confluent). However, I would like to know how to reclone H9c2.
Thank you!
Sooo.
1. You have a protocol to differentiate your line to myofibers. (I hope it is short and simple one - like fcs-withdrawal).
2. Divide your cells from mother culture at low numbers into 24-wells plates. Let is this happen into four 24-well plates.
3. Grow the four 24-well plates about 70% confluency (or lower, ie 50%). If it is sufficient feed them during this. Then split them well by well into a 96-well plate (about cell number to high enough to start diff quickly (please approximate it). You can use a multichannel pipette to move the cell samples but change the tips amongst "clones". Keep the original 24-well plates at low confluency after split as "subclones". Know the origin of the wells on this 96-well plate.
4. According to your protocol, differentiate the 96-well plate and check in in where (in which well) is proper myogenic differentiation happened. Where is - that is your renewed cell line(s). (Note: during this, passage the 24-well plates accordingly if it is sufficient).
5. Choose your best, and some backup, and grow them up from the mother 24-wells, and freeze them to stock. Than you can work further them but probably those cells are not the same than the originals.
That is a very practical approach. Differentiation of myoblast can be easily see on phase contrast, even you can calculate something - or even repeat the procedure to get more reliable data. Others might be can help more... I worked a little on this field. But i worked a lot of clones.
Gee, it was a long one. If there are a lot of proper-like clones you can use further the original cell line. I have no number for this - above 50% might be good.
Hi!
Thank you for the explanation. I think now I have an idea how to do the reclone. I have never work with clones before so I have couple following questions.
1. Could you please explain more about looking at the myoblast differentiation under phase contrast? From what I know, after differentiation, they will be elongated and have multi-nucleus. Am I right? Will just looking at them under the scope is good enough or should I stain with some specific fluorescent color?
2. So after figuring out the best well where it has best differentiation : non-differentiated cells ratio, I can trace back to the mother well in 24-well plate and make stock from that well. Could you please recommend the ratio of differentiated: non-differentiated cells that consider good?
Again, thank you so much for your response. It does help!
1. yes for phase contrast. if you work later with some fluo signal, it is better to check it too for the selected wells.
2. check your cell line current state. count this using few parallel samples. even repeat few times. use the system in which your experiment is planned (ie some fluo work on glass, do on that). this will be your starting point. I think if you reach some number over 50-60% for multi-nucleated, you do not need to subclone... but i do not know this exactly.
Note that this selection procedure above is not really subcloning. It just tries to isolate a part of your cell population which is more responsive for differentiation. I think i can not help more. Good luck!
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