Feb 06, 2026

Public workspaceLive Cell Quality Check – SGT Lab

This protocol is a draft, published without a DOI.
  • Yujun Feng, Ph.D.1
  • 1Spatial and Genome Technologies Lab, University of Illinois Chicago
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Protocol Citation: Yujun Feng, Ph.D. 2026. Live Cell Quality Check – SGT Lab. protocols.io https://protocols.io/view/live-cell-quality-check-sgt-lab-hjptb4mnp
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: January 02, 2026
Last Modified: February 06, 2026
Protocol Integer ID: 237011
Keywords: cell quality control, 3' assay, 5' assay, LUNA FX7, cell suspensions for 10x genomics platform, live cell quality check, essential checkpoint for experimental control, cell suspension, cell rna, quantification of cell viability, cell viability, 10x genomics platform, essential checkpoint, rna, scrna, robust quality control
Abstract
This protocol outlines a robust quality control (QC) procedure for preparing single-cell suspensions for 10x Genomics platforms. It details the quantification of cell viability, density, and the assessment of debris and clustering—factors that directly influence doublet rates and data noise. By establishing clear 'Go/No-Go' criteria and discussing the risks associated with low-quality inputs, this guide serves as an essential checkpoint for experimental control and successful single-cell RNA sequencing (scRNA-seq) outcomes
Guidelines
  • Mandatory QC: Always perform a comprehensive cell quality check immediately prior to library preparation. Never assume a sample's quality based on previous processing steps.
  • The "Stop" Rule: Establish a strict "Go/No-Go" threshold. If a sample fails to meet viability or purity benchmarks, do not proceed to microfluidic loading. The cost of a failed sequencing run far exceeds the cost of re-preparing a sample.
  • Pilot Study Recommendation: A "pilot" or "mock" cell count is strongly recommended for unfamiliar tissue types or new dissociation protocols. This allows for the optimization of the dissociation method without risking expensive 10x Genomics reagents.
  • Determine Loading Density: Use the pilot study to calculate the expected cell recovery and determine the exact volume required to hit your target cell capture number (e.g., 8,000 cells).
  • Day-of Validation: Even if a pilot study was successful, a real-time QC check is mandatory on the day of the experiment to account for day-to-day prep variability.
Materials
Troubleshooting
Test schedule and sample preparation guidance
Schedule test with user:
After consultation, if the user decides to apply:
a. 3' assay, including basic 3', 3'+Feature Barcode, 3' by On-Chip-Multiplexing (OCM)
b. 5' assay, including basic 5', 5'+Feature Barcode, 5; by OCM
Then, a cell quality check is mandatory.

*Pilot Study Recommendation: A "pilot" or "mock" cell count is strongly recommended for unfamiliar tissue types or new dissociation protocols. This allows for the optimization of the dissociation method without risking the use of expensive 10x Genomics reagents.
Please advise the user to schedule a date and submit an iLab request ('Test Count') at least one day prior to the experiment.
Sample preparation guidance for user:
Currently, SGT lab only receives ready-to-check cell samples. The user will need to work on the tissue digestion and cell sorting or enrichment, if any. At least on the day of the actual experiment, the user will also need to provide the cells in the appropriate buffer/ medium that pertains to the requirements outlined in the 10X Genomics protocols.
Here are the requirements for buffer/medium:
  1. Standard buffer: PBS with 0.1-0.5% Bovine Serum Albumin (BSA)
  2. Make sure to avoid:
1) EDTA(ethylenediaminetetraacetic acid),
2) sodium azide
3) DNAse.
They could impair the functions of enzymes in library preparation steps or the quality of cDNA. If these ingredients are necessary for preparation, please let the user inform SGT lab. Typical solution is to wash the cells 2-3 cycles with an appropriate buffer to minimize the conc. of unwanted ingredients. The cycle of wash also depends on the initial conc. before washing.
Recommended cell number, concentration and volume are:
1) >500k cells ( lower number could cause loss of cell pellet during centrifugation)
2) 1000-3000 cells/ μL
3) >300 μL (this is for passing through cell strainer)
This applies to 3'/ 3'-OCM/5'/5'-OCM assays.
Rule 1 may not be necessary if the sample has good quality and is ready to be loaded onto the chip.
Rule 3 may not be necessary if the sample has already been passed through a cell strainer at the user's lab.
Critical
Check cell quality using cell counter LUNA FX7
Determine whether centrifugation is needed for concentration
If the concentration of the sample received is lower than the recommended range, then centrifugation is needed prior to cell counting. Check with the user for the centrifugation force and time they used. The default method is 300g, 5 min, at 4 °C.
Fluorescence mode for viability check:
1. Add 2 μL AO/PI viability dye in a 1.5 mL Eppendorf centrifuge tube.
2. Filter the sample through a 70 μm or 30 μm cell strainer. Pipette the sample, ensuring no cell pellet at the bottom.
3. Take 18 μL of the sample and mix with the AO/PI dye in the tube.
4. Take 10 μL of the mixed sample and add it to one channel on the cell counting slide.
For each sample, use a new channel on the slide.
The sample should be put on ice throughout the counting process in real-test.
5. Turn on LUNA FX7. Check if the flash drive is in the USB port.
It will be used to save and transfer the result.
6. Select 'fluorescence cell counting' mode ( don't select 'advanced' option)
7. Click 'SETTINGS' Make sure the chamber area is selected that has samples added. Slide setting is '8Ch.'
8. Click 'PROTOCOL'. Here click 'mouse_fresh', or other protocol if needed. Then click 'LOAD'.
9. Click 'COUNT'.
10. Insert the slide. Chamber '1' is the entering side of slide. Then click 'INSERT' at the bottom right corner of screen.
11. Click 'COUNT'. Now the instrument will start autofocusing. Tube the focus level by clicking ∧ or ∨ key on the screen.
12. After focusing is finished, click 'COUNT'.
13. When counting is finished, name and save the result. Remember to select the saving location on the USB drive.
14. Share the data with user via BOX folder.

Critical
Typan blue mode for viability check (not recommended):
This dye should only be used if the sample is labeled with a dye that overlaps with acridine orange/propidium iodide (AO/PI). However, trypan blue is a less specific dye, which means it may stain non-cell debris and lead to an overestimation of cell numbers and quality when counted.
1. Add 10 μL Trypan Blue to a 1.5 mL Eppendorf centrifuge tube.
2. Filter the sample through a 70 μm or 30 μm cell strainer. Pipette the sample, ensuring no cell pellet at the bottom.
3. Take 10 μL of the sample and mix with the Trypan Blue dye in the tube.
4. Take 10 μL of the mixed sample and add it to one channel on the cell counting slide.
For each sample, use a new channel on the slide.
The sample should be put on ice throughout the counting process in the real test.
5. Turn on LUNA FX7. Check if the flash drive is in the USB port.
It will be used to save and transfer the result.
6. Select 'bright field cell counting.'
Steps 7-14 are the same as is in part <5>
Determine if the sample is qualified for the next step
1. Cell viability is >80% for cultured cells or >70% for primary cells. If viability is lower, the user will then decide whether to proceed to the next step. The quality of the data could not be guaranteed to a standard level.

2. Cell debris is low. Cell debris is defined as small pieces under bright field that are not stained with AO or PI. Most of the time, it will be below the size of smaller cells ( <~3 μm). Or It may have a linear shape, such as collagen. As a general rule, if the cell-to-debris ratio is less than ~1:1 (by a rough visual check), proceeding to the next step may be risky. The quality of the data could not be guaranteed to a standard level.

3. Cell clumping is minimal. Cell clump is defined as pieces that are stained with AO or PI, and can be clearly found as multiple cells under Bright Field. As a general rule, if the clump-to-single-cell ratio is more than ~0.05:1 (by a rough visual check), proceeding to the next step may be risky. The quality of the data could not be guaranteed to a standard level.
If not pass: troubleshooting
1. Cell viability low: live cell enrichment or sorting with low pressure could improve the viability.
2. Cell debris is high: centrifugation usually helps. If not, a debris removal kit might help. Sometimes cell debris level is correlated with cell viability. Dying cells tend to generate more cell debris.
3. Cell clumping is high: This usually requires optimization of digestion if using tissue samples, such as adding DNAse to remove sticky DNA components(be careful to wash thoroughly before scRNA-seq). If culture cells, using a longer time for dissociation might help. Or, use cell sorting to remove clumps.
If pass: guidance for next step
If this is a mock/pilot cell quality test, please verify with the customer the number of cells to input during the actual test.
Critical