I have a rare cell population and I am doing FACS sorting using cell surface marker staining to purify one cell type. I later plan on to pool the RNA from the same cell type from different animals. Since all animals are processed on different days but the cell type remains the same, Do I need to run the unstained sample always before running the stained sample for FACS OR once I can run the unstained, record the data and use it for other samples as well? This way I will save my sample and get more RNA.
Any help will be appreciated.
It is generally recommended to run an unstained sample as a control for each individual FACS experiment, regardless of whether or not the cell type is the same across different samples or animals. This is because there can be variability in the staining efficiency and purity of the cell population from experiment to experiment, even if the cell type and staining protocol are the same. Running an unstained control allows you to assess this variability and ensure that the staining is consistent across samples. If you want to save sample and get more RNA, you can consider pooling the unstained control samples from different experiments and use the data from a single control sample for multiple experiments.
Thanks Maria Arshad for your valuable answer, but pooling won't work for me as I will do experiments on 4 different days and FACS also needs to be done on all four days itself. I can't fix the cells and keep.
Running an unstained sample always helps you set the gating and gives you consistent data.
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