What are some best practices for culturing adherent and suspension cells with the 12-well Cytos™ Slide Kits on the AVITI24™ system?

Question

What are some best practices for culturing adherent and suspension cells with the 12-well Cytos Slide Kits on the AVITI24 system?

Answer

See the Cytos Sample Preparation & Optimization User Guide and the Cell Culture Protocols for use with Cytos Cell Screening for best practices for culturing suspension and adherent cells. These guides include protocols for surface coatings, seeding density, and provide images of successful cell culture techniques.

This article supplements the user guides with additional tips, videos, and images for key cell culture and sample preparation steps.

Summary

The following best practices apply to adherent and suspension cell workflows:

• Custom Surface Coatings
• Optimize Confluency Using Bright Field Microscopy
• Optimize Confluency Using Cytos Onboard Optimization Kit
• Pipetting Techniques
• Seed Cells
• Distribute Cells
• Add and Remove Media

Guidance specific to adherent or suspension cells is also included:

• Considerations for Adherent Cells
• Considerations for Suspension Cells

Considerations

Before performing a Cytos run, optimize the following for your cell type and experimental conditions:

• Surface coating
• Cell seeding density and growth time
• Effects of experimental treatments, if applicable

When you are ready to seed onto the 12-well Cytos Slide Kits, follow the protocols in the user guide and review the cell culture and fixation best practices described here.

Refer to the Knowledge Base article for modifications specific to the 48-well and 1-well Slide Kits.

Custom Surface Coatings

Cytos Slide Kits are available uncoated and require a surface coating. Test multiple surface coatings to ensure you are using the best coating for your cell type. See the Custom Surface Coatings section of the user guide for coating options and detailed instructions.

Optimize Confluency Using Bright Field Microscopy

See the Seeding Density Optimization and Assess Cell Sample Using Bright Field Microscopy sections of the user guide for detailed instructions. Additional recommendations include:

• Optimize seeding density in the 12-well Cytos Slide Kit. If additional optimization is required, use a 96-well glass plate as described in the user guide.
• Test a range of seeding densities and incubate cells according to your experimental needs.
• Use bright field microscopy as a quick and easy way to confirm that cells are evenly distributed and have reached the target confluency. See the Cell Culture Protocols and Cell Culture Hub for example images of the ideal confluency.

Optimizing Confluency Using Cytos Onboard Optimization Kit

See the Optimization Run section of the user guide for detailed instructions to use the Cytos Onboard Optimization Kit. Use this kit to ensure your cells are compatible with Cytos reagents and that your seeding densities are optimal for segmentation. Additional tips and recommendations include:

• Use the Cytos Onboard Optimization Kit to ensure cells are compatible with Cytos reagents.
• The Cytos Onboard Optimization Kits also allows you to image the cells labeled with core cell paint markers to check if your seeding densities are optimal for segmentation.
• After you have optimized your seeding density and timeline for ideal confluency, proceed to seeding into the 12-well Cytos Slide Kit for a Cytos protein or RNA detection assay.

Pipetting Techniques

• Proper pipetting techniques are critical to ensure an even distribution of cells in the slide kit and minimize damaging the slide surface. See the Pipette Placement Guidance in the user guide and the videos in the following sections.
• Use the slide gasket as a guide to avoid contacting the slide surface. Carefully move the pipette tip down the plastic well wall until you feel resistance from the gasket, then pipette slightly above the gasket.
• If poor cell distribution or image quality is observed after a run, see our Knowledge Base article on improving cell culture and cell screening images for troubleshooting guidance.

Cell Culture Best Practices

See the Sample Preparation section of the user guide for specific instructions for adherent cells and suspension cells. The following guidance supplements the user guide protocols:

Seed Cells

• If you are preparing a series of dilutions, prepare the dilutions in a deep-well plate.
• When possible, avoid adding cells and then topping up the well with additional media.
• When seeding cells, aim to add cell suspension into the slide kit in a single step (unless your cell line has special requirements). See the following video:
  

Distribute Cells

Follow the distribution procedure specified for your slide kit configuration and cell type in the Sample Preparation section of the user guide. Additional recommendations include:

• Place the slide in a 10 cm petri dish for easier handling. You can place up to four slide kits in a petri dish. See the diagram in the Sample Preparation section of the user guide.
• If using a petri dish, cover the slide kit with the petri dish lid rather than the flow cell lid while distributing cells.
• Distribute cells with a back-and-forth and side-to-side motion, as shown in the following video. Do not move in a circular motion.

Add and Remove Media

After you have seeded and attached the cells, use the following techniques for adding and removing media (see the user guide for specific volumes per slide kit format):

• Add media slowly along the middle of the well wall. This allows the liquid to flow more gently across the cells. See Pipetting Techniques.

• To remove media, tilt the slide and position the tip towards one corner, and avoid making contact with the slide surface. Follow the recommendations in the user guide to ensure you remove the appropriate volume at each step.

Considerations for Adherent Cells

See the Culture and Fix Adherent Cells section of the user guide for a detailed protocol, and the following additional tips for handling adherent cells:

Incubate Cells after Distribution

• Avoid rotational movement when transferring the slide kit to the incubator.
• After placing the slide kit in the incubator, repeat the distribution motion to help maintain even cell distribution.
• Incubate cells to a target confluency of 50–70% for most cell types. For example, we incubate HeLa cells for 16–18 hours.
• Contact Support for additional cell-line-specific guidance if needed.

Centrifuge Cells

• Most adherent cell workflows do not require centrifugation. However, all 48-well slide kits (regardless of cell workflow) need centrifugation to prevent bubbles from forming on the slide surface. See the recommendations for 48-well slide kits in the Knowledge Base and the Culture and Fix Adherent Cells section of the user guide for more details.

Fix Cells

See the Fix Cultured Adherent Cells section of the user guide for detailed instructions:

• Use the half-liquid exchange method: leave half of the liquid from the final wash, then add an equal volume of 8% formaldehyde to achieve a final concentration of 4% formaldehyde solution.
• Fix adherent cells at room temperature for 20–30 minutes.

Considerations for Suspension Cells

See Attach and Fix Suspension Cells section of the user guide for detailed instructions, and additional tips for handling suspension cells:

Centrifuge Cells

• All suspension cell workflows require a centrifugation step to attach them to the slide kit.
• Cover the wells with the slide kit lid before placing the slide kit in the Cytos Slide Kit Tray.
• If using one slide, place it in the center of the slide kit tray.
• Always balance the slide kit tray in a centrifuge with another slide kit tray.

Fix Cells

See Fix Attached Suspension Cells section of the user guide for detailed instructions and review the following tips:

• Suspension cells do not require washes before fixation.
• After centrifuging the slide kit, add an equal volume of 8% formaldehyde to achieve a final concentration of 4% formaldehyde solution.
• Fix suspension cells at room temperature for 20–30 minutes.

Additional Resources

For cell-line-specific instructions, see the following Cell Culture Protocols for use with Cytos Cell Screening:

• A549
• HeLa
• HepG2
• SH-SY5Y
• U2OS
• Jurkat