The successful isolation of nuclei is an essential step in both ATAC-sequencing workflows and single cell RNA-sequencing. Successful library preparation requires users to ensure that all sample input is non-clustered and debris-free.
Dual fluorescence counting using the CellDrop™ FL Automated Cell Counter is a useful tool, able to distinguish unlysed intact cells from isolated nuclei.
Cluster analysis software provides an aggregation estimate to the user, with the resulting images allowing the user to verify that any debris has been effectively removed.
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Counting nuclei with AO/PI stain
Acridine orange and propidium iodide (AO/PI) are utilized to confirm successful nuclei isolation. In conventional cell viability testing, the AO/PI dye combination will stain live cells so that these fluoresce green while staining dead cells so that these fluoresce red.
The staining process will also label any isolated nuclei red while labeling any remaining intact cells green. This additional feature allows the user to calculate any remaining intact cells as a percentage value of total counted cells.
This step is important in determining whether or not the experimental workflow can go ahead.
The nuclear pore complex permits passive diffusion up to 30-60 kDa, meaning that AO and PI (~0.6 kDa) are able to freely pass into the nucleus. This will lead to a red signal, which is the result of a FRET interaction between the two fluorophores.
It is vital that the number of intact cells in the isolation be minimized. Accurately enumerating intact cells using AO/PI can help improve quality control, as well as enhancing consistency of results in downstream workflows.
Nuclei isolation procedure and considerations
In the examples presented here, nuclei were isolated from HEK293T cell cultures in line with the 10X Genomics® protocol for the “Isolation of Nuclei from Single Cell Suspensions. CG000124 Rev D.”
Prior to lysis, cell density and viability were evaluated using the standard CellDrop AO/PI protocol, confirming a minimum of 2.5 million cells/mL at >90% viability.
Minimization of large clusters and debris is important in ensuring an effective downstream single cell sequencing workflow because these could clog fluidic chips and lead to low quality libraries or unsuccessful sequencing experiments.
Nuclei suspensions should be filtered to remove any cellular debris post-lysis. Should large clusters of nuclei be observed, it is recommended to refer to the manufacturer’s protocol.
It is also vital to remove intact cells that did not lyse during the procedure. Single cell sequencing procedures like those used by 10x Genomics® are reliant on isolated nuclei.
Without this, the technology may struggle to detect expression differences in a given cellular population.
Applications such as ATAC-seq necessitate the presence of intact nuclei in order to work correctly. Generally, sample volume is a limiting factor for these methods, so it is advisable to use a single analysis volume for multiple quality control purposes.
The unique DirectPipette™ Technology available with CellDrop Automated Cell Counters allows counting without the need for disposable slides, while the instrument’s variable chamber volume can accommodate counting volumes between 5 and 40 µL of sample.
CellDrop can also be used with common disposable plastic or even reusable slides, allowing users to quantify nuclear isolation on the CellDrop and then transfer the same slide to a microscope with higher magnification capabilities before performing an analysis of nuclear integrity.
Nuclei isolation AO/PI count protocol
The count protocol settings shown below were found to be accurate for both quantifying and qualifying nuclei isolated from multiple cell lines using the CellDrop AO/PI App. These cell lines included Jurkat, HEK293T (Figure 1) and CHO cells.
Table 1. Recommended Settings for Counting Isolated Nuclei. Source: DeNovix Inc.
| . |
| 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 |
Figure 1. Isolated HEK293T nuclei result image from the CellDrop. Nuclei are stained red while leftover intact cells are stained as green. Image Credit: DeNovix Inc.
Nuclei count and cluster size reporting
The data provided by CellDrop includes a count of intact cells versus nuclei, ideal for quality control purposes. Images are saved to the large onboard hard drive automatically, and these can either be inspected for debris using the large HD touch screen or exported via Ethernet, USB, or WiFi.
The instrument is also capable of generating comprehensive reports suitable for printing to network printers or saving as PDF documents.
Single cell sequencing technologies advise the use of lysing as a part of the sample preparation. This is to ensure that size limits for microfluidics systems are not exceeded.
In order to achieve this, CellDrop also reports cluster size (Figure 2), and this additional quality control step allows clusters to be excluded from analysis via the software’s size gating options.
Figure 2. Count cluster data from the CellDrop. When CellDrop counts cells/nuclei it also analyzes the result for clusters of objects. The software allows the user to visualize the cluster size and the frequency of up to six different cluster sizes for each count on the device. Image Credit: DeNovix Inc.
Counting nuclei with trypan blue stain
It is possible to analyze the success of a nuclear isolation using trypan blue (Figure 3), should dual fluorescence instrumentation be unavailable.
DeNovix recommends that fluorescent assays are used to quantify isolated nuclei wherever possible, however, because of the increased accuracy afforded by the obvious differences between green and red signals.
It is important to note that counting debris-laden samples via trypan blue has the potential to increase the number of erroneous counts, regardless of whether this is performed manually or with an automated cell counter.
Nuclei isolation trypan blue count protocol
A number of count protocol settings were confirmed as being accurate for nuclei isolated from multiple cell lines using the CellDrop Trypan Blue App.
These include Jurkat, HEK293T and CHO cells. Diameter adjustments may be required, however, depending upon the cell line under investigation.
Table 2. Recommended Settings for Counting Isolated Nuclei Using Trypan Blue. Source: DeNovix Inc.
| . |
| 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 |
Figure 3. Isolated nuclei counted with trypan blue on the CellDrop. The CellDrop does an excellent job of recognizing isolated nuclei in a carefully purified isolation of HEK293T cells. The nuclei are stained dark, indicating uptake of the trypan blue dye (circled red), while intact cells exclude the dye and remain bright (circled green). Image Credit: DeNovix Inc.
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