I am growing small cell lung cancer suspension cells. How do I separate dead cells from live cells while splitting the cells after confluency in flask?
Hi Chandra Kishore
* Using low speed centrifugation at around 500rpm for 10mins. This would allow the debris to stay afloat and the live cells to form a pellet. The time can be optimised according to the cell line.
Thanks,
Hello Chandra Kishore,
You may use density gradient centrifugation. In density gradient centrifugation depending on the density of the particles in the sample, similar substances will group together when exposed to rotational force. Because dead cells and cellular debris are fractured, they become less dense than living, healthy cells. Adding in certain separation reagents such as Ficoll can purify the sample by acting as a barrier that only one population can pass through.
For instance, in a 50 mL centrifuge tube, you may layer 18ml of your cell suspension onto 12ml of a Ficoll-paque combination. Centrifuge your tube for 15 minutes at 400x g. When centrifugation is complete, you will note that the live cells will collect at the interface and the dead ones will form a pellet at the bottom of the tube.
Another simple method as mentioned by Samir would include centrifuging the cell suspension at 150-200g for 10 mins. You may discard the supernatant which consists of cell debris and resuspend the cell pellet in fresh medium.
Best.
An efficient but more expensive approach would be to use a Dead Cell Removal Kit e.g.
https://www.miltenyibiotec.com/AT-en/products/dead-cell-removal-kit.html
https://www.stemcell.com/products/easysep-dead-cell-removal-annexin-v-kit.html
Centrifuge your suspension culture. The key is to keep it low and slow so as not to kill any live cells in the process. The standard is 400x g for 15 minutes
Use fluorescent dyes like propidium iodide (PI) to selectively stain dead cells. Analyze the cells using flow cytometry or fluorescence microscopy to distinguish between live and dead populations.
To separate dead cells from live cells when propagating suspension cells, you can use several methods. Here are a few options:
1. Density Gradient Centrifugation: This method utilizes the difference in density between live and dead cells to separate them. A density gradient medium, such as Ficoll or Percoll, is prepared, and the cell suspension is layered on top. Upon centrifugation, live cells float to the top of the gradient, while dead cells sink to the bottom. The specific protocol may vary depending on the cell type, so it is advisable to consult literature or established protocols for guidance.
2. Flow Cytometry: Flow cytometry is a powerful technique that can be used to discriminate between live and dead cells based on their fluorescence properties. Live cells can be stained with cell-permeant dyes, such as propidium iodide (PI) or 7-amino-actinomycin D (7-AAD), which are excluded by viable cells but can penetrate dead cells and label their DNA. By analyzing the cells using flow cytometry, you can differentiate between live and dead cells based on their staining patterns.
3. Trypan Blue Exclusion Assay: This simple viability assay involves staining the cells with Trypan Blue, which can enter dead cells with compromised membranes. Live cells exclude Trypan Blue and appear clear, while dead cells take up the dye and appear blue. By counting the stained (dead) and unstained (live) cells using a hemocytometer or an automated cell counter, you can estimate the viability of your cell population.
These methods can be used individually or in combination to effectively separate dead cells from live cells during propagation of suspension cells. The choice of method will depend on factors such as the cell type, the number of cells, and the resources available in your laboratory.
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