Please recommend an efficient method to reduce cell clumping in DLD1 cancer cells?

To minimize clumping, here are some suggestions:

Try using a lower concentration of trypsin when detaching the cells. A higher concentration of trypsin can lead to excessive cell detachment and increase the risk of clumping.

Gently pipette the cell suspension up and down to break up any clumps before plating the cells.

Pre-treat the cells with Accutase or TrypLE to detach the cells, which can reduce clumping compared to trypsin.

Try using a cell strainer to remove large clumps or cell aggregates from the suspension before plating the cells.

Consider using a cell dissociation buffer such as EDTA, which can help to prevent clumping.

Try using a low-attachment culture dish or plate, which can reduce cell aggregation and clumping.

When plating the cells, avoid pipetting the suspension too vigorously to prevent cell damage and clumping.

Malcolm Nobre

Hello Udeshi Wickramarachchi

Additionally, you may consider using DNase I at concentrations ranging from 20-100µg/ml to avoid formation of clumps. For each cell type, the working concentration must be determined individually.

Cell clumps form because of the presence of free DNA in cell suspension which are released from dead cells. Free DNA attract cells which bind altogether forming clumps.

The dead cells are formed when you usually subculture over-confluent cells. Also, over exposure of cells to trypsin during trypsinization can lead to cell death. So, subculture cells when they are 70-80% confluent at 1:3 to 1:10 i.e. seeding at 1-3 x 10,000 cells/cm², and during trypsinization expose cells to trypsin for not more than 5 mins.

Best.

Ahmad Al Khraisat

Clumping of DLD1 cells is a common problem encountered during cell culture, and it can be caused by various factors such as suboptimal culture conditions, cell density, and cell handling techniques. Here are some possible solutions to the clumping problem:

Try to optimize the cell culture conditions: Ensure that the cells are being cultured in a suitable medium with appropriate supplements and growth factors. Check the pH and osmolality of the medium and adjust as needed. Also, make sure that the cells are being cultured at the correct temperature and CO2 concentration.

Use enzymatic digestion to separate cell clusters: Try using an enzyme such as trypsin or Accutase to detach the cells from the culture dish. Use a minimal amount of enzyme and incubate for the minimum time required to avoid over-digestion.

Reduce the cell density: Lowering the cell density during seeding and passaging can help to reduce clumping. Try plating the cells at a lower density, and split the cells more frequently to prevent overgrowth.

Mechanical agitation: Try using a pipette to disperse the cells during seeding and passaging. Apply gentle pressure to the pipette to break up the cell clusters without damaging the cells.

Try to culture the cells as a single-cell suspension: If the above methods fail, try to culture the cells as a single-cell suspension. This can be achieved by using a cell strainer or a FACS machine to filter out the clusters before seeding.

Overall, optimizing the culture conditions, reducing cell density, using enzymatic digestion, and using mechanical agitation can all help to reduce clumping of DLD1 cells. It may take some trial and error to find the method that works best for your particular cell culture protocol.

Udeshi Wickramarachchi

Thank you very much Malcolm Nobre and Ahmad Al Khraisat . I reduced the trypsinization exposure in one of the cultures and it seems to help.

1. Another culture plate of the same DLD1 cells which has a confluence of 40-50%, the cells seem to have different types of morphologies. Some cells have clear margins and a shape while some look like dense areas. I want to know if this is normal with DLD1 cells or does this happen due to cell clumping and as a result cells not having enough space to grow.

2. Also, I need to treat these cells with a chemical before RNA isolation. Please recommend cat which stage the cells should be treated?

Ahmad Al Khraisat

Udeshi Wickramarachchi It is normal for cells to have different morphologies based on their growth stage and confluence. As cells approach confluence, they can start to pile up on each other, which can lead to different morphologies. Additionally, DLD1 cells are known to be heterogeneous, which means they can have different morphologies even at the same growth stage. However, if the cells are clumping and not having enough space to grow, this can affect their viability and proliferation. It is important to ensure that the cells are not overcrowded, and that the culture conditions are optimal for their growth.

The optimal time to treat cells with a chemical before RNA isolation depends on the specific chemical and its mode of action. Some chemicals may require longer incubation times to achieve maximum effect, while others may need to be added at specific time points during the cell cycle. It is best to consult the literature or manufacturer's instructions for the specific chemical you are using to determine the optimal time for treatment. Additionally, it is important to ensure that the treatment does not affect the integrity or quality of the RNA, as this can affect downstream analyses.

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