H1781 cells are shown to tend to clump each other.
H1781 is adherent cell and detached by using trypsin-EDTA.
however, over-digestion using trypsin might provoke cell clumping.
they seemed like big graph shape cell dough.
isn't there good way to separate cells as monocyte?
except for physically tapping way.
please, help me by suggesting any good idea.
I suggest trying to use icebox under the cell culture flask for few seconds and shake it so maybe this will help you to detach the cell without trypsin. I used this technique with the isolated monocytes. Good Luck.
I appreciated your kindness sincerely.
I will try to apply for your suggestion.
Dissociating clumping cells typically involves breaking apart cell aggregates or clumps into individual cells. Techniques vary depending on the cell type and the degree of clumping. Common methods include:Mechanical Disruption:Pipetting: Gently pipette the cell suspension up and down to break up clumps. Avoid vigorous pipetting to prevent cell damage.Using a Pasteur pipette with a narrowed tip can be more controlled.Enzymatic Treatment:Trypsin or other enzymes: Enzymes can be used to break down cell adhesion molecules and dissociate cell clumps. The duration of enzymatic treatment should be optimized for the specific cell type.Cell Strainer or Sieving:Pass the cell suspension through a cell strainer or mesh to separate clumps from individual cells.Dissociation Media:Use specialized dissociation media containing enzymes or chelating agents designed to break up cell clumps gently.Agitation:Gentle shaking or agitation may help disperse cell clumps. Avoid excessive force to prevent cell damage.
Hello Han yoon tak
First and foremost, you should avoid the formation of cell clumps. This can be done as follows.
1. Avoid over exposure of cells to trypsin. Over exposure to trypsin during trypsinization can lead to cell death. Cell clumps form in culture because of the presence of free DNA in cell suspension which are released from dead cells. Free DNA being sticky in nature attract cells and debris which bind altogether forming clumps. So, during trypsinization expose cells to trypsin for not more than 5 mins.
2. Avoid subculturing the cells when they are 100% confluent. Dead cells are formed when you usually subculture over-confluent cells resulting in the formation of clumps. So, subculture cells when they are 70-80% confluent.
3. You may follow the protocol given below for trypsinization.
Remove the medium and rinse with 0.25% trypsin, 0.03% EDTA solution. Remove the solution and add an additional 1 to 2 mL of trypsin-EDTA solution. Allow the flask to sit at room temperature (or at 37°C) until the cells detach. Add fresh culture medium, aspirate, and dispense into new culture flasks.
4. If you pellet cells too hard, they can clump. Ensure that cells are not subjected to high centrifugal force.
5. Use Ca++ and Mg++ free PBS as calcium and magnesium can promote cell clumping.
Removal of cell clumps:
In case the cell clumps are small, you may pipette the cell suspension a few times gently up and down to form single cell suspension. If you still fail, then calculate the volume of DNase I solution that should be added to the sample to yield a final concentration of 100 μg/mL DNase I. Add DNase I solution dropwise to the cell suspension while gently swirling the tube. Incubate at room temperature for 15 minutes. Wash the cells with culture medium or buffer containing 2% FBS. Gently invert to mix, then centrifuge at 300 x g for 10 minutes at room temperature. Discard as much of the supernatant as possible, then gently resuspend the pellet. If cells still appear clumpy, then you will have to pass the sample through a 37 - 70 µm cell strainer into a fresh conical tube. Rinse the sample tube three times with the culture medium or buffer containing 2% FBS, then pass through the strainer.
Best.
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