Hi Zubair,

For LMH cells, you should not use trypsin anymore as these are easily detached cells. Also, the normal pattern of LMH cells is like a patch, it will take some time till become confluent so increase the serum up to 15% and will be OK. If your cells are old and you used normal trypsin that will make it aged quickly, I would rather recommend to use low acting trypsin.

Best wishes my friend

AOA Rohaim

Hope you are fine.

Thanks for your response. I worked before on LMH with typical confluent monolayer using 10% media and IX trypsin. I am not sure why this is happening this time despite the fact that used approach and everything I am doing is same as before.

Regards

Hello,

Do not subculture the cells when they are 100% confluent but rather passage the cells when they are 80% confluent. Over confluent cells causes cell lysis and thereby release DNA. DNA being sticky in nature can cause cells to stick to each other forming clumps.

Also, avoid over exposure of cells to trypsin. Trypsinization for 5 minutes should be more than sufficient.

Check whether the cells are being exposed to stress such as contamination, changes in composition of the medium like change of FBS brand or lot number, pH changes in culture medium, etc.

There must be something which you must have missed out.

Hope this is helpful.

Good Luck.

Thanks Malcolm

I first give a quick wash with 1 ml trypsin just like we do with PBS (1X). After that, I add 0.5 ml trypsin in 25cm flask and let it flow unless it covers the cell surface. The cells start detaching within 30 seconds and therefore I do expose them with trypsin for less than a minute. The moment cells are going off the surface, trypsin is taken out and 10% media is added along with pipetting in a way that one can see single cells under microscope. However, when I put them in incubator and visualize after 20-30 minutes, I see formation of chains first followed by clumps and cells shows growth restricted to patches.

Do you use LMH cells too now? If so, can I have step-by-step procedure you are using for passaging LMH cells.

Regards

For everyone who may read this thread in future regarding LMH. I figured out the way and it worked very well with LMH cells. A ste-by-step appriach is given:

Best of luck!

Muhammad,

There is no need to take out the trypsin after the trypsinization step. The FBS in the media that you add is inactivating the trypsin. 5% FBS/trypsin is the inactivation proportion. In 6mL of 10% FBS media there are 0.6mL of FBS, more than enough to inactivate the 0.5mL of trypsin.

Keep them cold, precool the glass slides for microscopy too

They may be low cell concentration in distribution, or low serum concentration. Try to add L glutamine in your media.

Thanks everyone for your comments and suggestion. However, I found rinsing cells with Trypsin-EDTA solution twice followed by cell incubation for next 2-3 minutes the best approach for sub-culturing.

Regards