Question (29972) | Lysosomal Staining Kit - Blue Fluorescence - Cytopainter (ab112135)

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Question

 Hi there,  I bought your CytoPainter Lysosomal Staining Kit - Blue Fluorescence (ab112135) and I have tried it on MCF7 cells for multi-color labeling experiment. However, I could not counter-stain nucleus with Hoest 33342 after staining with your lysosomal staining kit, and your lysosomal staining kit emit significant background green fluorescence which is detrimental to my experiment.  May I know what is your advice using your kit to perform multi-color labeling experiment? How to reduce the background green fluorescence emission? I incubated my cells in your working solution for 30 minutes then I change the staining solution into PBS containing Hoest33342 and mitotracker red and incubate for another 15 minutes. Thanks.

Answer

Thank you very much for your inquiry. I am sorry to hear you have been experiencing problems with the CytoPainter Lysosomal Staining Kit. Due to the wide emission spectrum property of the CytoPainter dye it is normal to also have some green fluorescence; some of our customers actually like this property for using their violet filter sets. Currently, a narrow spectrum of the blue lysosomal dye is being developed and might be available soon. In terms of the multi-color labeling, due to potential interference of the compounds and overlapping fluorescence spectra we would not recommend this particular combination, i.e. using blue for the lysosome detection, red for mitochondria, and Hoechst33342 which also emits in the blue spectrum (for excitation/emission spectra details see for example here: http://www.mcb.arizona.edu/ipc/fret/index.html). We would recommend to either use a different dye for live cell DNA staining, or otherwise if you prefer to use Hoechst33342 for DNA staining to use the CytoPainter Lysosomal Staining Kit in green for staining lysosome (ab112136). If you need to avoid any green signal, a redesign of the experimental layout might be required. There are for example DNA dyes (such as DRAQ5 and DRAQ7) available that emit in the far red or orange spectrum, which would require a different fluorescence for the mitochondria. For both mitochondria and lysosomal staining we have different colours available. I am sorry that I could not be of more help on this occasion and hope this information is nevertheless helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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