BioStatus been working with the BD FACSDiscover S8 at a leading SRL and training site on the use of DRAQ5™, as described in the article that described this novel instrument's capabilities.
In the course of this work it was quickly determined that the production model, not surprisingly, significantly outperforms the experimental platform utilised in that original Science journal article (1).
Central to this, sensitivity is markedly different. We determined that the optimal concentration for DRAQ5 is 0.5 - 1.0 µM for a suspension of 1x10⁶ cells/ml.
Therefore, DRAQ5 will be the preferred choice in most applications since at this concentration it is highly economical and delivers desirable and well-defined nuclear labeling in the far-red. This fluorescence property releases the two other fluorescence channels for further biological parameters.
DRAQ5 is already validated in many hundreds of imaging flow cytometry papers using the Imagestream X mark II (CyTEK Biosciences) (2) and its common use in preparative single cell /nuclei sorts for transcriptomic and genomic analyses (3). These are two techniques that, for example, the BD FACSDiscover S8 is able to combine.
Importantly, all users of DRAQ5 can be assured of its quality and consistency maintained over many years of experience at BioStatus, and the global availability of the product through carefully selected channel partners or, as always, direct from BioStatus. (Buy DRAQ5 here)
NOTE: We recommend the BioStatus product SKU: DR05500 (500 µl; 500 µM) for convenient pipetting of between 1 and 2 µl directly into each ml of cell suspension (to achieve a 0.5 -1.0 µM final concentration, as above). This product is ideally formulated for use on the BD FACSDiscover S8 and is available direct from BioStatus.
It should be borne in mind that the concentration maybe somewhat different for different cell types but it is unlikely to vary by more than 2-3 fold, given the experience of the 12,000+ citations for DRAQ5 in the published literature, across a very broad array of human cells.
These concentrations fall even further below those already determined to be entirely compatible with downstream RT-PCR and PCR amplifications, as described here.
Also, it should be possible to use the related DNA dye CyTRAK Orange™, with the caveat that this has peak emission at 610 nm with a differential staining of nucleus and cytoplasm (shown to be segmentable by intensity masking and a watershed, respectively, in microscopy). We have not tested CyTRAK Orange yet on the FACSDiscover S8 but would predict that it might require a concentration of 1 - 2 µM, based on experience of a wide variety of cell analysis platforms and from what we have already learned about DRAQ5 on the FACSDiscover S8.
This story will be expanded on in the coming months.
References:
1. Schraivogel D, Kuhn TM, Rauscher B, Rodríguez-Martínez M, Paulsen M, Owsley K, Middlebrook A, Tischer C, Ramasz B, Ordoñez-Rueda D, Dees M. High-speed fluorescence image–enabled cell sorting. Science. 2022 Jan 21;375(6578):315-20.
2. Harte DS, Lynch AM, Verma J, Rees P, Filby A, Wills JW, Johnson GE. A multi-biomarker micronucleus assay using imaging flow cytometry. Archives of Toxicology. 2024 Jul 12:1-7.
3. Ordoñez‐Rueda D, Baying B, Pavlinic D, Alessandri L, Yeboah Y, Landry JJ, Calogero R, Benes V, Paulsen M. Apoptotic Cell Exclusion and Bias‐Free Single‐Cell Selection Are Important Quality Control Requirements for Successful Single‐Cell Sequencing Applications. Cytometry Part A. 2020 Feb;97(2):156-67.
BD FACSDiscover S8 and BD are trademarks of Becton, Dickinson and Company
© Copyright BioStatus Limited 2024
No comments :
Post a Comment