Good morning,
We have started to work with PANC-1 and we have faced problems in cell counting. We count less cells than what we really have. We use DMEM + 10% FBS + 1% Penicillin/Streptomycin medium and we use trypsin 0.5% with EDTA without calcium and withouy magnesium (https://www.biowest.net/media/l0930c.pdf). After adding trypsin, we incubate at 37ºC during 5-10 minutes (we also have tried during 15 minutes) and then we centrifugate it 1500 rpm during 5 minutes. We count cells with trypan blue and automatic cell counter. The problem is that we can see aggregation of cells in the cell counter and for this reason we think the cell counter is not able to count it properly.
We have tried to disagregate cells with a better homogenization technique, increasing the trypsin time incubation and with colagenase but we have not succeed.
There is anyone who have faced the same problem and can provide us some ideas to try to improve our cell count?
Thanks in advanced.
Hello Paz Moreno
Environmental stress can accelerate the rate of cell death, resulting in the release of DNA molecules that are sticky from the dying cells. The sticky DNA molecules clump neighboring cells together.
Trypsin concentration in 1x working solution can range from 0.025–0.5%, depending on trypsin activity or potency, incubation time and the type of cells. If you are not sure about the concentration of trypsin to use, start with a low concentration. Avoid using too high concentration of trypsin or exposing the cells to trypsin for too long a time. Exposing the cells to trypsin for 15 mins could damage the cells and even kill them.
Before using trypsin/EDTA, you may rinse the cells with PBS (without Ca2+/Mg2+) and give a brief rinse (few secs) with prewarmed trypsin/EDTA. Then expose the cells to prewarmed trypsin/EDTA solution for not more than 5 mins.
In case you fail to reduce cell clumping, you may treat the cell suspension with DNase I. Add a final concentration which should be 100ug/ml DNase I to the cell suspension. Add DNase I solution drop wise to the cell suspension while swirling the tube gently. Then incubate the tube with the cell suspension at room temperature for 10 minutes.
Wash the cells with culture medium or buffer containing 1%FBS, centrifuge, discard supernatant and resuspend the pellet.
Hope this helps.
Best.
Hello Malcolm Nobre, thanks for your answer. I'll try.
I've other question. My panc-1 cultures are so dirty. It is not contamination. Do you think is it normal??
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