How do I assemble a Cytos™ flow cell?

Question

How do I assemble a Cytos flow cell?

Answer

Before starting a Cytos run, you need to assemble the slide into a flow cell using components from the AVITI24™ Flow Cell Assembly Kit. The Assemble the Cytos Flow Cell section in the Cytos Sample Preparation & Optimization User Guide provides step-by-step instructions. This article provides tips, videos, and photos of key steps for additional help and context.

Summary

Assembling a Cytos flow cell requires the following major steps:

• Disassemble the Slide Kit
• Align the Slides
• Seal the Slides
• Assemble the Flow Cell Cassette
• Clean the Assembled Flow Cell
• Maintain the Flow Cell Sealer

This article provides additional tips, images, and videos for each of these major steps.

Considerations

This article outlines best practices for flow cell assembly using the AVITI24 Flow Cell Aligner and Sealer, which is included in the AVITI24 Flow Cell Assembly Kit. See the Cytos Sample Preparation & Optimization User Guide for detailed step-by-step instructions.

Disassemble the Slide Kit

The Disassemble the Slide Kit section in the user guide provides detailed instructions for disassembling the slide kit. Consider the following additional tips, images, and videos:

• Dry slides properly: Appropriate drying of slides is essential. Using slides that are not completely dry before flow cell assembly increases the risk of bubble formation on the slide. However, flow cells that are left to dry for too long before a run (> ~10 minutes) can cause a reduction in protein performance. Examples of wet and dry slides are shown in Figure 1.

Figure 1. (A) Example of a wet Cytos slide. (B) Example of a dry Cytos slide

• Aspiration: Drying the slide can require multiple aspirations, or you can use a multichannel aspirator. See the following video:

• Use pull tabs: You can disassemble the slide kit using plastic pull tabs, as shown in the following video:

• Dry after disassembly: Slides can also be dried after disassembling the slide kit if needed. The edges of the slide should be free of liquid so that a clean seal is created with the adhesive slide when sandwiching. See the following video for an example:

• If unsure, skip aspiration: If you don’t feel confident that you can aspirate liquid from the slide without touching the cells, proceed with flow cell assembly without additional liquid removal.

Align the Slides

The Align and Seal the Slides section in the user guide provides detailed instructions to align the slides in the AVITI24 Flow Cell Aligner. Consider the following additional tips, images, and videos:

• Clean the aligner: Before seating the slide, ensure the Flow Cell Aligner is clean by briefly wiping it with an isopropyl wipe. See Maintain the Flow Cell Sealer for details.
• Seat both sides: Slides need to be properly seated on the sample side and the adhesive side of the aligner.
• Sample slide placement: The following video shows the placement and alignment of the sample slide into the aligner:

• Check slide seating: Figure 2 shows an example of a poorly seated sample slide (A) and a properly seated sample slide (B) in the aligner:
  • A poorly seated slide has edges that are lifted above the recessed area, and the slide is not fully clamped in place, as in Figure 2A.

Figure 2. Tips for aligning the flow cell. (A) Example of a poorly seeded slide. (B) Example of a properly seeded slide.

• • A properly seated slide is flush with the recessed area and all pins are properly engaged to clamp the slide in place, as in Figure 2B.
• Remove the adhesive cover: After the slides are properly seated on the sample side and the adhesive side of the aligner, remove the adhesive cover on the adhesive slide as shown in the following video:

• Sandwich the slides: The Align and Seal the Slides section in the user guide provides detailed instructions for sandwiching the sample and adhesive slides in the aligner, as shown in the following video:

• Check aligner contact: After closing the aligner, inspect and confirm it has made complete contact. Figure 3 shows an example of poor aligner contact (A) and proper aligner contact (B).
  • Poor contact occurs when the aligner is not perfectly flat, which you can see by the raised left post in Figure 3A.

Figure 3. Tips for alignment. (A) Example of a sealer with poor contact. (B) Example of a sealer making proper contact.

• • Proper contact occurs when the top of the aligner is completely parallel to the bottom and you can see contact between the top and bottom parts of the aligner along the entire edge, as in Figure 3B.

• Release the slides: After opening the aligner as shown in the following video, press the button on the adhesive side to release the sandwiched slides:

Seal the Slides

The Align and Seal the Slides section in the user guide provides detailed instructions for sealing the sandwiched slides with the AVITI24 Flow Cell Sealer. Consider the following additional tips, images, and videos:

• Check the slide is flat: After placing the slide in the sealer, you can run your fingers along the recessed area of the sealer to ensure the slide is lying flat with no lifted edges.
• Figure 4 shows an example of a poorly seated slide from two different angles. The slide is not fully in the recessed area and has one or more edges lifted

Figure 4. Examples of poorly seated sandwiched slide in the Sealer. (A) The slide is lifted on the left side and not in the fully recessed area. (B) The slide is lifted along the top edge.

• Sealing motion: For an example of the sealing motion with a properly seated slide, see the following video:

• Check flipped slide: When you flip the slide over and repeat the sealing process, always check that the slide is lying flat with no edges lifted before moving the roller grip.

Assemble the Flow Cell Cassette

The Assemble the Flow Cell Cassette section in the user guide provides detailed instructions for assembling the flow cell cassette.

Clean the Assembled Flow Cell

Cleaning the assembled flow cell is critical for achieving high-quality images. Contaminants on the outer surface of the flow cell can impact the Cytos run. The Clean the Assembled Flow Cell section in the user guide provides detailed instructions for cleaning the assembled flow cell. Consider the following tips:

• Use fresh gloves: Always use fresh gloves. Carryover from reagents or pens can result in autofluorescence or out-of-focus objects.
• Wipe with isopropyl: Wipe both the top and bottom slides with a clean isopropyl wipe. Quickly follow up with a lens wipe to remove excess isopropyl. Allowing the isopropyl to dry on its own can create a film that causes background fluorescence.
• Use canned air carefully: Canned air can be used to remove any additional dust or fibers that have settled on the flow cell. Firmly grasp the flow cell and hold the canned air upright to avoid introducing additional contaminants. If handled improperly, canned air can leave propellant liquid on the flow cell surface.

See also the Knowledge Base article on Out-of-Focus Objects in Cytos runs for more details.

Maintain the Flow Cell Sealer

The Maintain the Flow Cell Sealer section in the user guide provides detailed instructions for maintenance and storage of the AVITI24 Flow Cell Sealer. Additional tips, images, and videos are provided here:

• Remove the roller grip for cleaning: If a slide has been damaged on the sealer, remove the roller grip to facilitate cleaning, as shown in the following video:

• Clean the sealer: Clean the sealer with isopropyl wipes, as shown in the following video: