Introduction
Isolating nuclei is a critical step for single cell RNA-sequencing and ATAC-sequencing workflows. Ensuring that non-clustered, debris free samples are input is crucial to successful library preparation. Dual fluorescence counting using the CellDrop™ FL Automated Cell Counter is used to distinguish unlysed intact cells from successfully isolated nuclei. Cluster analysis software provides the user with an aggregation estimate, and the resulting images allow the user to determine if the debris has been successfully removed.
Counting Nuclei with AO/PI Stain
Acridine Orange and Propidium Iodide (AO/PI) are used to determine the success of a 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 carryover as a percent of 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 will display a red signal due to a FRET interaction between the two fluorophores. Minimizing the number of intact cells in the 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 Procedure & Considerations
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.
Minimizing debris and large clusters is important for the downstream workflow of single-cell sequencing, since these can clog the fluidic chips resulting in low quality libraries or failed sequencing experiments. It is therefore critical for nuclei suspensions to be filtered to remove cellular debris post-lysis. 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. Alternatively, CellDrop is also compatible with common disposable plastic or reusable slides. This can 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.
Nuclei Isolation AO/PI Count Protocol
The following count protocol settings were determined to be accurate for quantifying and qualifying nuclei isolated from multiple cell lines including Jurkat, HEK293T (Figure 1), and CHO cells using the CellDrop AO/PI App.
Table 1: Recommended Settings for Counting Isolated Nuclei| Count Application | AO/PI |
| Chamber Height | 100 µm |
| Dilution Factor | 2 |
| Diameter(min) | 4 µm |
| Diameter (max) | 20 µm |
| Live Roundness | 1 |
| Dead Roundness | 1 |
| Green Fluorescence Threshold | 10 |
| Red Fluorescence Threshold | 1 |
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 the large HD touch screen or exported using WiFi, Ethernet or USB. Comprehensive reports can also be generated for printing to network printers or saving 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 2) 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 3). DeNovix recommends the use of fluorescent assays for quantifying isolated nuclei where possible, due to the increased accuracy insured 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 Protocol
Count protocol settings determined to be accurate for nuclei isolated from multiple cell lines including Jurkat, HEK293T, and CHO cells using the CellDrop Trypan Blue App. Diameter adjustments may need to be made depending upon the cell line.
Table 2: Recommended Settings for Counting Isolated Nuclei Using Trypan Blue| Count Application | Trypan Blue |
| Chamber Height | 100 µm |
| Dilution Factor | 2 |
| Diameter(min) | 6 µm |
| Diameter (max) | 30 µm |
| Live Roundness | 50 |
| Dead Roundness | 15 |
| Stained Threshold | 15 |
Revised 24 Nov 2020
10x Genomics® is a registered trademark of 10x Genomics, Inc.
