Reagent formulation and dispensing with CELLSTUDIO Software on the ICELL8 cx system

• Best practices for dispensing reagents
• Reagent test parameters
• Additive tables

ICELL8 cx CELLSTUDIO v2.6 Software (CELLSTUDIO Software) allows researchers to innovate and develop new applications for the ICELL8 cx Single-Cell System. The system's open format gives users greater control of cell selection, reagent formulation, and reagent and sample dispensing for downstream processing.

Developing new applications may involve dispensing novel reagents that need to be tested to ensure they have minimal cross-contamination and can be precisely dispensed prior to being incorporated into a workflow. To assist with this process, Takara Bio scientists have generated guidelines for reagent formulation and dispensing. We have tested commonly used additives such as Polyethylene Glycol 8000 (PEG 8000), glycerol, Triton X-100, NP-40, Tween-20, and SDS to address these concerns.

The guidelines below are intended to help researchers save time in their application development efforts without compromising confidence in their data.

When to use these guidelines

These guidelines are only necessary when developing your own custom applications for the ICELL8 cx Single-Cell System using CELLSTUDIO Software. The ICELL8 cx Single-Cell System User Manual (Appendix G, "Designing an Application: Advice from Our Scientists") notes when these guidelines are applicable.

Blank ICELL8 cx chips and additional consumables which should be used during custom application development and usage are available for order from their product page.

If you're an ICELL8 cx Instrument user and haven't yet upgraded to CELLSTUDIO Software, you can request an upgrade from Technical Support or have it upgraded during an onsite service visit. Purchases of an ICELL8 cx Instrument in 2020 or later will have CELLSTUDIO Software preinstalled.

Best practices for dispensing reagents

1. When dispensing a reagent that has a high concentration of detergent and/or viscous additives, it is best to observe how cleanly the reagent dispenses. A user can observe the blotting paper to see how much of the liquid dispensed was on top of the chip. Ideally, there should be little to moderate blotted area on the paper.
   
   

   

   Figure 1. Examples of blotting paper illustrating a range of dispense quality. These images can be used as a reference for your dispenses to help evaluate the quality. Left image. The red circles indicate where spots were found. The number and small size indicate a good dispense. Center image. The blue circles again mark where damp spots were found. Slightly larger, this dispense is considered within acceptable parameters. Right image. With this much liquid absorbed by the paper, the dispense would be considered 'poor'.

2. After the dispense, users should use [Stream Check] (under the Advanced tab of CELLSTUDIO Software) to flush out the system lines. Following the stream check, users should perform 2–3 tip clean cycles. Performing an alcohol prime is also recommended if the tips still do not seem clean. See the user manual for the steps on how to do these two processes.

Reagent test parameters

All reagents used in testing were at room temperature.

Average dispense volume test

We measured the density of the reagent using a scale that goes to 0.0001 g, sensitive enough to accurately measure the volume dispensed. We dispensed the reagent into an Single-Cell 250f Chip (Cat. # 640193) and recorded the weight before and after blotting the chip. Using the measured density, we calculated the average dispense volume. We considered the dispense successful if there was no large area of liquid blotted off the chip, and the average dispense volume was within 10% of the expected volume.

Satellite droplet test

We prepared reagents with a 1:200 dilution of a UV-reactive dye. We dispensed onto Single-Cell chips in a checkerboard pattern and imaged the chips afterward on a SmartChip ND Real-Time PCR Cycler. We counted the instances of satellite droplets on top of the chip that can pose a risk for contamination. We considered the dispense to have passed if the total number of satellite droplets was less than 3–4% of the total number of dispensed wells.

Test guidelines

We have provided two sets of concentration ranges based on these tests. Users can decide what concentration range to follow when dispensing based on their concerns.

Passing concentrations

• We define "minimum passing concentration" as the lowest concentration that was tested during this study and passed the criteria established for the given test. We have not tested concentrations lower than this limit. While dispensing a reagent below this concentration limit may be fine, the user would do so at their own risk.
• We define "maximum passing concentration" as the highest concentration that was tested during this study and passed the criteria established for the given test. In most cases, we also tested concentrations higher than this limit, and they failed the criteria established in the protocol. We would not recommend users go beyond the maximum passing concentration benchmark.

Summary

When formulating reagents for a novel in-chip application on CELLSTUDIO Software, users should determine how compatible the additive concentrations are with the ICELL8 cx system. An ideal in-chip dispense would have limited cross-contamination between wells and accurate dispense volumes. We have taken these concerns into consideration when developing this application note. Users can follow the concentration ranges established from this study to facilitate a smoother transition from in-tube reactions to in-chip reactions without having to frequently reformulate the reagents.