One other new flow cytometery information drop from Oliver Burton, simply published in Current Protocols.
This one is on optimising blocking whereas preserving sign, specifically the best way to overcome Fc interactions and dye-dye interactions, and stopping tandem break-down.
The small print are within the protocol, with variants for intracellluar and cytokine staining, however generally-speaking regular mouse/rat serum, BioLegend Tandem stabilizer and Thermo/BD Good Stain Buffer is an optimum combo. True-Stain and different additions aren’t value the additional $$$.
For Tandem Sign Enhancers, you do not really want them for mouse cells, and for human cells a less expensive different is just to repair your cells and stain with tandems after fixation. Each eliminates non-specific tandem binding and in addition reduces tandem break-down. Since monocyte blocks scale back transcription issue detection, for some cause, depart them out if you’re doing intracellular staining.
The Good and Tremendous Shiny dyes actually do want the Good Stain buffers, however bear in mind that these buffers are mildly fluorescent, so depart them out in the event you do not want the dye, and titrate them down while you use them. 1/2 to 1/4 is generally good, and for a lot of antibodies even decrease is okay (and cheaper!).
For the tandem dyes, Tandem Stabilizer is sweet, however you may make it simpler by way of panel design. Tandem breakdown isn’t purely chemical – it’s larger on monocytes than lymphocytes, and is essentially abolished in fastened cells. So transfer these tandem dyes to post-fix T cells in the event you can!
We have tried to cowl all the primary use instances, so check out Oliver’s trouble-shooting guide to cut back off-target binding and protect sign.
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