Cell Culture Hub
Analyze the before-and-after AVITI24™ run images to identify optimal cell confluency for better experimental outcomes.
AVITI24 onboard imaging to be used as an example of confluency
Overview
The Cell Culture Hub guides AVITI24 users in optimizing cell culture parameters, such as seeding density and surface coatings, in a plate-based format before preparing samples for a Cytos run. Cytos users can compare their bright field and AVITI24 images with Cell Culture Hub images from a similar preparation to assess cell confluency and adjust culture parameters for optimal results. For users using different cell types than those listed, reference the cell line with the closest diameter and same growth conditions (i.e., adherent or suspension) for ideal confluency comparisons. Users can also reference the Cytos Cell Culture Protocol webpage for specific protocols and images. The approximate diameter of cell lines used in these experiments is listed below:
| Cell | Size |
|---|---|
| Hela | ~30-35 μm |
| Jurkat | ~10-15 μm |
| SH-SY5Y | ~18-25 μm |
The About the Experiment section explains how these images were generated. This optimization protocol is recommended for users who are new to Cytos or are working with unvalidated cell types. For optimization and sample preparation protocol details, refer to the Cytos Sample Preparation & Optimization User Guide.
About the Experiment: Suspension - Jurkat
Surface Coating:
Prior to cell culture steps, CellVis 96-well plates (Cellvis catalog # P96-1.5P), Glass-bottom 96-well plates (Ibidi catalog # 89627), and 12-well slide kits were custom coated with Poly-L-Lysine (PLL) as detailed in the Cytos Sample Preparation & Optimization User Guide.
Cell Culturing:
- Jurkat cells were cultured with complete RPMI-1640 in a stationary T225 flask to ensure sufficient cell count for the optimization experiment.
- Cells were gently pipette-mixed to break any clumps that may form in the culture during growth.
- Cells were counted using a hemocytometer to determine the approximate cell concentration in the culture.
- An aliquot containing the appropriate number of cells was taken from the culture flask. The cells were centrifuged at 200 x g for 5 minutes.
- The cell pellet was resuspended in 1x PBS to an appropriate concentration for the highest cell concentration. The cell suspension can be recounted to confirm starting concentration for optimization titration.
Cell Seeding Optimization:
- For the cell concentration titration, each concentration was diluted individually with 1x PBS in an intermediate tube before seeding into the plate and slide kit wells. This ensures a uniform cell suspension and even distribution of the culture during seeding.
- Each concentration was seeded in replicates across both the plate and slide kit. The seeding volumes are listed below for each configuration used:
| Vendor | Layout | Well Shape | Well Area | Seeding Volume per Well |
|---|---|---|---|---|
| CellVis | 96-well Plate | Circle | 30 mm² | 100 µL |
| Ibidi | 96-well Plate | Square | 7.4 mm x 7.4 mm | 150 µL |
| Element Biosciences | 12-well Slide Kit | Squircle | 7 mm x 7 mm | 150 µL |
The seeding concentrations, listed as cells per well in 150µL seeding volume, for both the 96-well plate and 12-well slide kit are listed below:
| Row | 96-well plate | 12-well Slide Kit |
|---|---|---|
| A | 500,000 | 300,000 |
| B | 300,000 | 300,000 |
| C | 240,000 | 120,000 |
| D | 180,000 | 120,000 |
| E | 120,000 | 30,000 |
| F | 60,000 | 30,000 |
| G | 30,000 | -- |
| H | 10,000 | -- |
- Cells were seeded and fixed to the surface as described in the Cytos Sample Preparation & Optimization User Guide for suspension cells.
Visual Assessment
Cells were visualized either through brightfield microscopy (40X magnification) or on instrument. On-instrument visualization allows for visualization of cell paint markers to additionally assess background, segmentation, and provides quantitative output such as mean intensity and localization ratio. Preparation for on-instrument visualization can be found in the following user guides:
- Cytos Protein User Guide
- Cytos 3′ Transcriptome User Guide
- Cytos Optical Pooled Screening User Guide
- Cytos Multimodal Pooled Screening User Guide
If you have any questions on how to seed your cells for your experiment, contact Element Support.