I've done hanging drop with A549 cells. (10 ul/20.000 cell/drop) At second day I saw a few cells have attacht on lid, behind the sphere colony. At third day they become much more cells that attachtes on lid. Does anyone have ever experienced like that? How can they attacht on lid? 1) I don't think there is no Poly-D-Lysine on lid and 2) How did they withstand gravity?
I'd expect the cells would attacht each other and going down because of gravity but some of them going up and attacht on lid.
I used to do a lot of hanging drop culture of murine ES cells but never noticed any cells attaching to the lid. Is anyone slamming your incubator door?
Hello Okan.
If you leave too long to make too many drops, the early drops would show such attachment and then proper crawling on the on lid. The other points are that you should have nearly a half ball shape of cell-containing microdrops in the least region of plastic lid with great care to avoid flattened and spread dropping, and too many cells in 10ul drop.
Regards.
Thank you Mr. Hrstka. I don't think so. Just me and one of my colleagues are use incubator.
Thank you for your time Mr. Lee. I'm not sure if I understand you right. What does it mean "leave to long to make too many drops"? I pippette 10 ul drop on lid that is contain 20.000 cells each drop. Just that. I did not do anything to drops after that. Just observed and taking images.
Hello Okan.
I corrected typographical error, now. What I meant was that the intervals between making the first drop and the last drop is too long due to your intention to make many hanging drops on the lid. Say, 1 min or 2 min that is enough time for the highly packed cell suspension to allow to touch down on the lid under lamina flow steering forces would make a typhoon to the cells in the 10ul drop, evaporating quickly, too. All this physical factors could generate cell attachment. In addition, consistent whirling effects generated by fanning in incubator would make further opportunity for cells to touch to the roof lid specially in the flattened and packed drop with high number of cells.
Other possibility is the prolonged observation and image recording since the microdrop evaporate so quickly, leading to hyperosmotic and acidic conditions.
Open your mind to all the possibilities mentioned above. "Trust me, I am a doctor" in hanging drops of mES cells, hES cells, MCF7 cells and other many embryonic cells isolated from fast growing tissues including cancer.
Regards.
Certainly the possibility of evaporation leading to shrinking drops is a good suggestion. Are you keeping a liquid in the bottom of the plate for humidification?
Increase drop size to 20-22 microliter, that way the cells will be hanging, as you have been adding to many cells/drop with only 10ul.
Hi,
Try to use special low attachment culture dishes.
Good luck!
I've always assumed all lids to be "low attachment," as I doubt any manufacturer would bother treating the lids.
To get ultra-low attachment plates are cover by special Ultra-Low Attachment surface coating. The plates which haven't been covered with the special surface are characterized by high attachment potency.
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