The Nuclei Trypan Blue (left) and Nuclei AO/PI (right) apps on the CellDrop Automated Cell Counter are optimized with protocols for isolated nuclei counts.
Keep reading to learn how to count isolated nuclei on the CellDrop.
Nuclei Isolation Procedure & Considerations
As a proof of concept, nuclei were isolated from HEK293T cell cultures according to the 10X Genomics® protocol for the “Isolation of Nuclei from Single Cell Suspensions. CG000124 Rev D.” Before lysis, cell density and viability were assessed using the standard CellDrop AO/PI protocol to confirm a minimum of ~2.5 million cells/mL at >90% viability.
With the basic performance established, isolations were performed from fresh and frozen tissue using Miltenyi Biotec’s Nuclei Extraction Buffer (130-128-024) and gentleMACs™ Dissociator. Images in this technical note have been selected from mouse liver samples, but extractions have been tested on mouse frozen mouse lung, prostate, kidney, brain, and liver, fresh mouse prostate, brain, and liver, and frozen rat lung.
Keeping the nuclei at 4°C throughout the protocol is important for high quality extractions. To ensure high yields, it is recommended to keep samples on fresh ice and work as quickly as possible. While not necessary for high yields, the gentleMACS™ Octo Coolers (130-130-533) were used in this procedure to keep nuclei at optimal temperatures throughout the extraction process.
The resulting images showed a high extraction efficiency (>90%), but also contained cellular debris as is expected of whole organ isolations. While the gentleMACs™ Dissociator system and filtration steps reduce debris, different tissue types may be more challenging to work with. Tissues from the brain or heart, for example, typically contain more debris than liver and lung samples. An optional step is to use Miltenyi’s Anti-Nuclei Microbeads (130-132-997) to clean up all the debris and leave only nuclei behind (see Figure 1). This demonstrates how high resolution images provided by the CellDrop enable visualization of samples to assess the amount of debris present that could interfere with downstream protocols.
Minimizing debris and large clusters is important for the downstream workflow of single-cell sequencing; these can clog the fluidic chips resulting in low quality libraries or failed sequencing experiments. Refer to the manufacturer’s protocol if large clusters of nuclei are observed.
Similarly, removing intact cells that did not lyse during the procedure is also critical. Single-cell sequencing procedures such as those employed by 10X Genomics® rely on isolated nuclei for the technology to appropriately detect expression differences in a cellular population.
Many applications such as ATAC-seq require intact nuclei for the technique to work properly. Typically the sample volume for such methods is a limiting factor, so using a single analysis volume for multiple quality control purposes can be beneficial. The unique DirectPipette™ technology of CellDrop Automated Cell Counters enables counting without disposable slides and the variable chamber volume allows counting volumes of between 5 and 40 µL of sample. In addition, CellDrop is also compatible with common disposable plastic or reusable slides to allow the user to both quantify the nuclear isolation on the CellDrop and transfer the same slide to a microscope with a higher magnification for nuclear integrity analysis.
Counting Nuclei with AO/PI Stain
Acridine Orange and Propidium Iodide (AO/PI) are used to determine the success of nuclei isolation. In traditional cell viability testing, the AO/PI dye combination stains live cells so they fluoresce green and dead cells fluoresce red. However, the stain will also label successfully isolated nuclei red and any remaining intact cells green. This allows the user to calculate the residual intact cells that carry over as a percent of the total counted and determine if the experimental workflow can proceed.
As the nuclear pore complex will allow passive diffusion up to 30-60 kDa, both AO and PI (~0.6 kDa) freely pass into the nucleus and display a red signal due to a FRET interaction between the two fluorophores. Minimizing the number of intact cells in isolation is important, so accurately enumerating the intact cells with AO/PI can improve quality control and improve consistency in the results of downstream workflows.
Nuclei Isolation AO/PI Count
Figure 1 was made using the CellDrop Nuclei AO/PI App to analyze nuclei isolated from mouse brain tissue, and Miltenyi’s Anti-Nuclei Microbeads were used to remove debris. Figure 2 depicts a CellDrop result image of nuclei isolated from HEK293T cells. The CellDrop quantifies isolated nuclei (stained red) and the intact cells (stained green) to calculate the nuclei extraction efficiency for each sample. This data is readily presented to the user on the results screen with other relevant sample data.
Nuclei Count and Cluster Size Reporting
CellDrop data provides the user with a count of intact cells vs. nuclei for quality control purposes. All images are automatically saved to the large onboard hard drive and can be inspected for debris using the large HD touchscreen or exported using Wi-Fi, Ethernet, or USB. Comprehensive reports can also be generated for printing from network printers or saved as PDF.
Single-cell sequencing technologies recommend lysing as a part of the sample prep to ensure size limits for microfluidics systems are not exceeded. CellDrop reports cluster size (Figure 3) enabling an additional quality control step. Clusters can be excluded from analysis using size gating options available in the software.
Counting Nuclei with Trypan Blue Stain
Where dual fluorescence instrumentation is not available, it is also possible to analyze the success of a nuclear isolation using trypan blue (Figure 4). DeNovix recommends using fluorescent assays for quantifying isolated nuclei where possible due to the increased accuracy ensured by the clear differences in green and red signals. It should be noted that counting debris-laden samples using trypan blue can increase the number of erroneous counts either with an automated cell counter or by manual count.
Nuclei Isolation Trypan Blue Count
The following image (Figure 4) was taken in the CellDrop Trypan Blue Nuclei App during a measurement of HEK293T cells. The trypan blue dye enters all of the successfully extracted nuclei (circled in red) and stains them, giving them a dark appearance. The intact cells (circled in green) that were not successfully lysed exclude the trypan blue dye and appear white.
The numbers of nuclei and intact cells are used to quantify the extraction efficiency for a sample to ensure that enough nuclei are available for the subsequent experiments.
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gentleMACS™ Dissociator and gentleMACS™ Octo Coolers are registered trademarks of Miltenyi Biotec, and are used for identification and references purposes only. DeNovix, DeNovix products, and this website are not endorsed or authorized by, or in any way affiliated with Miltenyi Biotec.