Isolating nuclei is essential for single nuclear workflows like ATAC-sequencing and RNA-sequencing. Obtaining non-clustered, debris-free samples is important for successful library preparation. Even when debris does not directly impact library preparation, it is crucial to accurately count nuclei for single-cell analysis.
The CellDrop™ FL Automated Cell Counter facilitates dual fluorescence counting, enabling the distinct differentiation of nuclei, unlysed intact cells, and debris via a simple Acridine Orange/Propidium Iodide (AO/PI) assay.
Image Credit: DeNovix Inc.
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Nuclei isolation procedure and considerations
Nuclei were isolated from HEK293T cell cultures following the 10X Genomics® protocol for the “Isolation of Nuclei from Single Cell Suspensions. CG000124 Rev D.” Cell density and viability were evaluated before lysis using the standard CellDrop AO/PI protocol to confirm a minimum of 2.5 million cells/mL at >90% viability.
Reducing debris and large clusters is critical for the subsequent steps in single-cell sequencing workflows, as they can obstruct fluidic chips, leading to low-quality libraries or unsuccessful sequencing experiments.
Nuclei suspensions must, therefore, be filtered to eliminate cellular debris after lysis. Consult the manufacturer’s protocol if large clusters of nuclei are detected.
Ensuring the removal of intact cells that did not lyse during the procedure is also essential. Single-cell sequencing processes, like those utilized by 10X Genomics®, depend on isolated nuclei to accurately detect expression variations within a cellular population.
Numerous applications, including ATAC-seq, necessitate intact nuclei for the technique to work correctly. The sample volume for such methods is typically a limiting factor, making it advantageous to utilize a single analysis volume for multiple quality control purposes.
CellDrop Automated Cell Counter
The innovative DirectPipette™ technology of CellDrop Automated Cell Counters facilitates counting without the need for disposable slides, and the variable chamber volume enables counting volumes of between 5 and 40 μL of sample.
The CellDrop is also compatible with common disposable plastic or reusable slides. This enables users to quantify the nuclear isolation on the CellDrop and transfer the same slide to a microscope with a higher magnification for nuclear integrity evaluation.
The CellDrop has saved millions of plastic slides from disposal. Image Credit: DeNovix Inc.
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About DeNovix, Inc.
DeNovix Inc. is an instrumentation company that designs, manufactures and sells laboratory equipment to meet the demands of today’s evolving life science technologies. Our focus is on providing innovative products and outstanding customer support. DeNovix is equipped with the financial, commercial and technical resources to deliver breakthrough products for your research success.
Our multi-award winning products include the DS-11 Series Spectrophotometer / Fluorometer and CellDrop™ Automated Cell Counter. Each instrument is a fully networkable, stand-alone system with an HD touchscreen and custom EasyApps™ software designed by life scientists for life scientists. Labs love the smart-phone-like operation, impressive performance, and the flexible connectivity of the instrument.
Learn more about DeNovix instruments and how they can optimize your research workflows.
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