Aplo Flow

Welcome to ONI’s comprehensive Aplo Flow user guide. This guide is intended as a reference manual for the unboxing, installation, operation, and maintenance of both the Aplo Flow PREP System and Fluidics Driver. Additional assay-specific sample preparation guides are available in conjunction with this guide.

Before using the system be sure to read this user manual to ensure proper operation and that all safety warnings are followed. Do not use any accessories or workflows that are not specified by ONI or are not in line with your local safety requirements. Maintenance should only be performed by ONI. ONI cannot provide support for issues arising from improper use or the use of non-approved workflows and/or accessories.

Improper use of the instrument could result in injuries or hazards, primarily electrical and biological hazards.

• Do not use the instrument if the lid sensor is not functioning. There is a high risk of injury from rapidly moving parts.
• Electric currents are running when the instrument is connected to line power. Do not open covers or remove parts, as this may damage the instrument or expose electric wires.
• The power supply must be connected to an outlet with appropriate grounding means.
• Do not replace the detachable main supply cord.
• Only use the instrument with the lid down.
• Do not move the Aplo Flow system during operation.
• Always transport the Aplo Flow system with travel locks applied and in the original shipping container. Failure to apply travel locks and using incorrect shipping containers when moving the Aplo Flow system will void your warranty. Travel locks should be removed before powering the instruments to prevent damage.

Definition of terms, abbreviations, and acronyms used throughout this guide.

Aplo Flow is designed as a reliable automation solution for both sample preparation and complex assays requiring precise control of fluidic delivery in coordination with data acquisition. ONI Application Kits™ have been designed and formatted to enable unattended sample preparation of up to four (4) custom fluidic chips on the Aplo Flow system. A separate fluidics driver enables full automation of fluid delivery, exchanges, and logical construction of previously hard-to-automate assays.

Key Applications:

• Sample preparation: Automated sample preparation for ONI’s Application Kits™
• Lights out operation: assays can run throughout the night, unattended
• Fixed cell assays: such as Exchange PAINT with virtually unlimited plexity
• Live-cell assays: such as particle uptake tracking or drug delivery studies

The Aplo Flow is intended for research use only (RUO) and is not approved for diagnostic or clinical applications.

• Place all equipment on a clean, dust-free, and flat benchtop surface.
• Keep all objects and obstructions at least 15cm from the back of the instrument and any air vents.
• Avoid installation locations which suffer from temperature fluctuations e.g. under air vents and windows with periods of direct sunlight.
• Avoid locations close to water sources, e.g., sinks and water baths.

Aplo Flow is designed for indoor use only.

Unboxing

The installation and setting up of the Aplo Flow PREP system should take around 30-60 minutes. ONI recommends that two people remove the instrument from its box: One to lift the instrument out and one to hold the box in place.

There are two layers to the packaging. The top ‘Accessory layer” that holds the accessories and cables, and the bottom layer containing the system itself.

Accessory layer

This layer contains several items which should be unpacked and placed aside for use later during setup.

Once the top accessory layer has been removed, the system below will be accessible to complete installation.

Status indicator color coding:

The first step of installation after unboxing is removal of the travel locks that protect the machine from damage during shipping.

Remove pipette head travel lock

1. Carefully loosen the screw on the pipette head travel lock by rotating it counterclockwise.
2. Pull the travel lock straight out towards the front of the instrument.
3. Thread the pipette head travel lock into its designated storage location on the back left-hand side of the deck.

Install the pipette head
The pipette head is shipped in a separate small box held securely in the first layer of the Aplo Flow shipping box. Installation is simple and requires no tools.

Unboxing

The installation and setup of the Fluidics Driver should take 30-60 minutes. ONI recommends that two people remove the instrument from its box: One to lift the instrument out and one to hold the box in place.

Similar to the PREP system, there are two layers to the packaging. The top ‘Accessory layer” that holds the accessories and cables, and the bottom layer containing the system itself.

Accessory layer

This layer contains several items which should be unpacked and placed aside for use later during setup.

System layer

Once the top accessory layer has been removed, the system below will be accessible to complete the installation.

There are four cutouts in the lower foam, which allow the system to be held from the bottom when lifting it out of the shipping box.

Hardware overview

Status indicator color coding:

The Aplo Flow system includes consumables (both single-use and semi-consumable) designed to specifically enable fluidics assays.

Individual consumables can be purchased from your local ONI representative following the SKUs below.

Fluid Path

The total Aplo Flow fluid path is comprised of the Cell Flow Chip combined with three labware components (tether, wash chip, and well insert ). Should any leaking be observed at the tether-to-chip connector, tubing, or well insert interfaces, a replacement assembly can be purchased by contacting your local ONI representative to purchase a full fluid path replacement kit.

Cell Flow Chip

A single-use, four (4) lane microfluidic chip, named Cell Flow chip, that supports live cell growth and automated fluidics. The fifth lane within the chip allows for manual addition of Tetraspeck beads for use in automated channel mapping. The Cell Flow chip comes pre-assembled as a chip ’sandwich‘ containing the Cell Flow Chip, a pipette adapter, and a lid, all contained in sterile packaging.

The Aplo Flow system has been tested and verified for compatibility with the following labware, which can be purchased directly from the vendors listed below.

1. Open the storage compartment on the back of the Aplo Scope by pressing the blue release button found inside the Aplo Scope sample area and by removing the rear lid.
2. With one hand firmly holding the release levers on the tether head, gently detach the tether head from the Wash chip (see images below from here on).
3. Connect the tether head to a Cell Flow Chip in the designated holding area on the storage plate.
4. Carefully pass the connected Cell Flow Chip through the fluidics passthrough at an angle.
5. Lower the front edge of the Cell Flow Chip under the tabs on the front of the fluidics stage retention clip.
6. Using an index finger and thumb on each side of the stage, pinch the back of the Cell Flow Chip so it snaps into place, flush with the sample stage
7. Finally, place the large retention magnets into their designated place on the sides of the Cell Flow Chip to hold it down securely.

Cross section of fluid path

The fluidic path is designed to manage excess flow through as the flow cell is washed with bulk volumes of reagents. The cell flow chamber outlet leads to a vertical overflow chimney that drains into a larger output well to catch flow through when washing the lane or seeding cells. Each output well holds 150 µL total.

Above each fluidic lane, a gas-permeable membrane separates the cell growth area from a fluidic reservoir. The reservoir is designed to prevent evaporation and maintain osmotic conditions to support healthy cell growth.

Manually seeding cells or reagents

1. Remove the assembled Cell Flow Chip from its sterile packaging and carefully set the lid aside when ready to transfer liquids.
2. Firmly press the tip of a pipette into the input side of the Pipette Adapter and slowly inject liquid into the flow lane. As liquid passes through the flow lane, excess should be visible rising from the overflow chimney and waterfalling into the output well.
3. Finally, add 100 µL of fresh media to the top of the gas exchange membrane before replacing the lid.

This section provides general guidance on flushing, cleaning, and the chemical compatibility of the fluidics path.

Flushing the system

Every fluidic assay should begin with a priming sequence of approximately 1.5 mL per lane (DI water, PBS, or the appropriate assay buffer) to displace air and ensure accurate delivery of fluids by the syringe system.

After an assay, flushing with de-ionized or MilliQ water is typically sufficient to clean the fluidic path. However, for more thorough cleaning or to remove any contaminating residue, it is acceptable to flush each lane of the full fluidic path either 70% EtOH, low concentration NaOH (0.1M), or dilute bleach (≤10%). Do not use solutions containing high concentration bleach or organic solvents. Do not leave any such reagents standing in the fluid path after cleaning—always end a session by following the short-term storage instruction below.

Short-term storage

For short term storage between assays (≤ 2 weeks), it is advised to flush the tether immediately after an assay concludes to reduce the risk of salt or biological build up in the fluidic path:

1. Start by attaching the tether head to the Wash chip and stowing it in the storage location in the back of the Aplo Scope.
2. Flush each lane of the fluidic path with 10-20 mL of de-ionized or MilliQ water.

Long-term storage

For extended storage between assays (> 2 weeks):

1. First, with the Wash chip connected, prime each lane with 10-20 mL de-ionized or MilliQ water.
2. Then fully prime each lane with > 30 mL of air per lane to purge all liquid from the fluidic paths.
3. Finally, disconnect the tether from the Wash chip and allow all fluidic paths to air dry completely.

Wetted materials

The following materials may contact fluid in the Aplo Flow path. When considering chemical compatibility, generally, contact time is as important as concentration; as short exposures followed by a flush are far less damaging than stagnant contact. For additional details on chemical compatibility, refer to Appendix 1. Chemical Compatibility.

Do not use

The following should not be introduced into the fluidic path:

• Concentrated bleach or other sodium hypochlorite solutions
• Acetone and other ketones
• Chlorinated solvents (chloroform, dichloromethane)
• Aromatic solvents (toluene, xylene)
• Concentrated strong acids and strong bases

The fluidics driver is designed to work with the included bottle and custom cap to prevent any hazardous liquid waste leaks.

To empty the waste bottle, follow the simple steps:

1. Hold the waste bottle near the lower half.
2. Pull the bottom of the bottle away from the fluidics driver at an angle.
3. Pull down and out, away from the fluidics driver, to release the bottle top from the waste cannula.
4. Remove the cap and dispose of liquid waste following applicable Environment, Health & Safety guidelines.
5. To reinstall the waste bottle for the next assay, simply reverse steps 1-3.

The following are current best-practice recommendations.

Minor bubbles

If a bubble is observed in the imaging area during acquisition, advance it out of the field of view by pushing one column volume (lane volume, 5–10 µL) at the standard flow rate. The Cell Flow Chip channel depth is 400 µm, so most small bubbles will easily flush out of the imaging area.

Persistent or larger bubbles

If bubbles repeatedly obstruct imaging—visible in the field of view or causing Z-lock timeout errors—flush the lane with wash buffer at 100 µL/s (20× the standard rate). After clearing the bubble, reload the target reagent following the standard procedure.

The Aplo Flow system is verified for standard operation at 5 µL/s. Higher flow rates have been tested up to 50 µL/s without issue, though detailed data is pending publication.

Counterintuitively, a faster flow rate can prevent bubble-induced cell detachment of live cells. Therefore, for live cell assays, a flow rate of 100–150 µL/s is strongly recommended. Every cell line and experiment is unique, so it is recommended to optimize the flow rate for each experiment to the specific cell line and reagents expected in each assay or application.

Due to elastic components in the fluidic path and the open-air pipettor side of the system, a brief stabilization period is required whenever flow starts or stops:

• At standard flow rate (5 µL/s): wait 3–5 seconds
• At elevated flow rates (100–125 µL/s): wait 5–10 seconds

Air gaps are recommended to prevent cross-contamination between adjacent reagents in the fluidic line.

• Recommended range: 10–50 µL per air gap
• Rationale: With an air gap >10 µL, the effective flush factor is ~5×. Without an air gap, the flush factor increases to ~60×, significantly increasing reagent consumption

Default: The example Python scripts use 50 µL air gaps for all reagent deliveries

To ensure that the target reagent reaches the imaging area at the correct concentration (accounting for bubble mitigation and the flush factor),the following minimums should be followed:

• Minimum volume per reagent: 150 µL
• Staging principle: The first 100 µL of each reagent passes through the lane (flush factor), with the remaining 50 µL staged within the sample area

Please refer to Appendix 2. Reagent delivery design for more detailed implementation details and assay design recommendations.

The Aplo Flow system is designed for years of trouble-free operation. To ensure optimal performance, some parts are designed to be serviced based on level of utilization or annually during preventative maintenance.

Replacement of the filter is recommended on an annual interval or sooner depending on air quality and conditions unique to each lab space.

Each Aplo Flow pipette head is calibrated from the factory for optimal performance. It is recommended that the pipette head undergo re-calibration on an annual basis to maintain peak accuracy and precision.

Reach out to your local field service engineer or sales representative to learn more about service offerings and the recommended Aplo Flow preventative maintenance schedule.

Designed for up to 250 uses before replacement is necessary. Specific lifespan of semi-consumable reagents may be shorter. The extent of useful life is based on reagents used and adherence to recommended cleaning procedures.

For issues related to the Aplo Flow PREP System or Fluidics Driver, please refer to the dedicated Aplo Flow Troubleshooting Guide or contact your local ONI representative for assistance.

• ONI resource library
• Aplo Scope Webinar
• Service Desk

At ONI, supporting our users is a priority. For the latest services and support information visit https://desk.zoho.eu/portal/onidesk/en/home or email us at support@oni.bio. All new equipment purchases will be covered by a one-year warranty.

• For general inquiries and questions please follow this link
• To contact a sales representative please follow this link

Ratings reflect the wetted materials in the fluidic path as intended to support reagent selection, not to guarantee performance in a specific application.  Compatibility depends on chemical concentration, temperature, and exposure time. A reagent that is acceptable for brief contact may degrade a material under prolonged or stagnant exposure. The user is responsible for validating compatibility for their intended reagents and operating conditions before use. When a reagent is not listed, evaluate it against the wetted materials table below and the published compatibility data for each material.

Ratings apply to the fluidic path as a whole and are set by the limiting material identified in each row.

Common reagents used in supported research applications are summarized below.

The total dead volume between the input well of the well insert and the Cell Flow Chip lane is approximately 450 µL.

When considering actual dead volume of the system there are two functional approaches to consider:

Approach 1 — Sequential (one-at-a-time) delivery: Each reagent (minimum 150 µL) is loaded individually into the pipettor and pushed to the chip using wash buffer equal to the dead volume. This eliminates the need for precise push volume calculations.

Approach 2 — Pre-loaded (queued) delivery: Multiple reagents are pre-loaded into the fluidic line, separated by air gaps, and delivered sequentially in a single operation. This approach requires precise push volume calculations to ensure accurate reagent positioning at each stage.

The loading_imaging_buffer function implements Approach 2 by default. The same function can be adapted for Approach 1 by replacing all steps after the first reagent and its air gap with equivalent volumes of wash buffer (push buffer).

Pre-loaded delivery staging example (loading_imaging_buffer):

Upon completion, the final 50 µL of each reagent is staged within the sample area. Subsequent reagents in the queue can then be advanced with a simple 200 µL push. Exact push volumes may vary depending on air gap size, presence of wash buffer between reagents, and actual loading volumes. Users are encouraged to refer to the delivery diagram and calculate push volumes independently.