Question
What are some best practices for cell culture and sample preparation in the 48-Well and 1-Well Cytos Slide Kits on the AVITI24™ system?
Answer
The Cytos 48-Well and 1-Well Slide Kits each require a few modifications during sample preparation compared to the 12-well slide kits. For example, they can require centrifugation after seeding and distributing cells, or use different wash volumes. These steps are detailed in tables for the affected steps in the Cytos Sample Preparation and Optimization User Guide.
Summary
This article provides cell culture and sample preparation best practices for Cytos 48-Well and 1-Well Slide Kits during the sample preparation workflow:
Considerations
The Cytos Sample Preparation and Optimization User Guide provides sample preparation details using each of the slide kit configurations (48-well, 12-well, and 1-well). This article highlights some of the modifications and general tips for handling the 48-well and 1-well slide kits. Always consult the user guide for detailed instructions for your kit.
48-Well Cytos Slide Kits
Consult the Sample Preparation section in the user guide for detailed instructions for adherent or suspension cells with each slide kit configuration. Use the following tips and videos for additional help:
Using Proper Pipetting Techniques
- Proper pipetting techniques are critical to ensure an even distribution of cells on the slide kit. See the Pipette Placement Guidance in the user guide and the following videos to further highlight these techniques.
- Use a 16-channel Finnpipette or a standard P200 multichannel pipette with a 48-well slide kit.
- If using a P200 to add or remove liquid, you may need to perform the motions twice or more per column depending on the volume.
- Contacting the slide surface can result in loss of cells. Refer to the Knowledge Base article on improving cell culture techniques for optimal cell screening images to see how contacting the slide surface affects Cytos images.
- To avoid scratching the slide surface, use the gasket as a guide to avoid contacting the slide surface. Carefully move the tip down the plastic well wall until you feel resistance from the gasket, then pipette slightly above the gasket along the well wall.
- When adding liquid, visually confirm that all pipette tips make contact with the well wall. This provides a smoother release of liquid and is less likely to dislodge lightly adhered cells.
- When you remove liquid from the slide, tilt the slide kit gently so that liquid pools to one corner and pipette from that corner, avoiding contact with the slide surface. Ensure some liquid remains in the wells to avoid cells over-drying.
Seeding Cells
- When dispensing cells, ensure that the slide kit is on a flat surface, and the pipette is perpendicular to the glass slide surface.
- Add cells into the center of the well in a smooth motion and avoid dropwise distribution or contact with the slide surface.
Distributing Cells
- For even distribution of cells across the wells, cover the slide kit. Gently distribute the cells for 30 seconds using a forward-and-back, pause for 5 seconds, and then side-to-side motion. as indicated in the Sample Preparation section of the user guide. You can also distribute cells with a rocker or shaker if desired.
- Move the plate with slide kits at a slow pace and ensure you are moving at least 5 inches in each direction, as shown in the video:
Centrifuging Cells
- Due to the shape of the wells in the 48-well slide kits, a centrifugation step after dispensing cells is required to ensure there are no bubbles and all liquid touches the glass surface.
- Omitting the centrifugation step can lead to inefficient cell adherence due to bubbles (Figure 1).
Figure 1. Example of insufficient cell adherence due to bubbles on the slide surface.
Figure 2. (A) Example of poor seeding with bubbles in many wells. (B) Example of correct cell seeding after centrifugation.
1-Well Cytos Slide Kits
Consult the Sample Preparation section in the user guide for detailed instructions for adherent or suspension cells with the 1-well slide kit. For specific guidance related to suspension cells with 1-well slide kits, see the step-by-step guidance to attach suspension cells on a 1-well slide kit. Use the following tips and videos for additional guidance:
Using Proper Pipetting Techniques
- Proper pipetting techniques are critical to ensure an even distribution of cells in the slide kit. See the Pipette Placement Guidance in the user guide and each of the videos below to further highlight these techniques.
- The 1-well slide kits require larger pipetting volumes. You can use a P1000 pipette or a multichannel P200 pipette to achieve an even distribution of cells.
- When adding liquid, always ensure that the liquid completely covers the well area. Pipette into opposite corners of the wells as shown in the following video:
Seeding Cells
- If using a P1000 pipette to seed cells, pipette along the length of the well to distribute cells.
- When adding media, rotate the slide to ensure media has fully covered the well.
Distributing Cells
- Distribute cells in a 1-well slide kit using the same forward-and-back, then side-to-side motion as indicated in the Sample Preparation section of the user guide. Consult the user guide for specific guidance for adherent or suspension cells.
- Take extra care to avoid splashing due to the larger well size. You can also distribute cells with a rocker or shaker if desired.
Centrifuging Cells
- Allow suspension cells to settle in the 1-well for 15 minutes before centrifugation.
- Only use the middle position in the slide kit tray when using the 1-well to ensure the best distribution.