I would like to investigate the effect of treatments on the cell surface expression of a cell line. Assuming that one treatment upregulates FITC-stained marker and the level of PE-stained marker remains the same relative to the vehicle control, do I need to do separate sets of compensation for the two fluorophores on the treated group as well as on the vehicle control before I could proceed with acquiring the results. I have this question in mind because I read about the leakage of one fluorochrome to another channel can be enhanced by an increase of its intensity. Since each treatment affects the fluorochrome intensity differently, does it mean that I need to do one compensation for every treatment, including the control? Thanks
Hi Yuan
Compensation needs to be set for each channel FL1 and FL2. The most appropriate is a positive control of your cell line of interest with a high level of either FITC or PE label. You are correct that as FIT brightness increase you will also see a more noticeable increase in PE bleed through. Though in experience I have not had more than 25% FL1 into FL2 and 5% FL2 into FL1 with those labels. If you do not have a suitable cell line control with sufficient brightness you can use labeled beeds as an alternative. Once compensation is set for the day those values do not change as you move from batch to batch. The most important thing is to use a control that represents the most brightest possible option. Also make sure that your negative compensation control is not measuring below the limit of detection. I hope this helps.
Steve
To calculate compensation, you can also use beads with yours antibody.
- Badges
- Science method