All of our cell line products are monoclonal cell lines.

According to our current company policy:

- With a signed Non-Disclosure Agreement (NDA), we are authorized to share the vector map.

- Without a signed NDA: We may disclose the sequence of the overexpressed target gene. Additionally, we can provide lentiviral residual testing reports upon request.

The following confidential information is not disclosed:

1. The complete vector sequence and the original vector name.

2. Sequences of signal response elements related to signal transduction pathways.

Cell lines are shipped on dry ice. To ensure optimal cell viability, we recommend thawing and initiating culture immediately upon receipt. If immediate thawing and culturing are not possible, we advise transferring the cells to liquid nitrogen for long-term storage. Please ensure the transfer process is quick to avoid thawing, as this may impact the long-term stability and viability of the cells.

If immediate transfer to liquid nitrogen is not feasible, the cells can be temporarily stored in a -80°C freezer. However, we recommend that the storage period from the date of receipt should not exceed two weeks. Long-term storage on dry ice or in a -80°C freezer is not recommended.

When using the cells, please refer to the recommended thawing and culturing methods provided in the DS.

Please Note: If the cells are received unfrozen or not on dry ice, please contact our technical support team immediately at techsupport@acrobiosystems.com.

We provide two cryovials of cell lines to ensure the smooth progress of your experiments. In the event of any issues with thawing, recovery, or culturing of the first vial, please contact our technical support team (techsupport@acrobiosystems.com) for troubleshooting before thawing the second vial.

All ACRO cell lines undergo pre-shipment validation for recovery and culturing. Additionally, we recommend establishing a cell bank at the earliest possible passage stage to ensure long-term use.

After thawing the cells, they should initially be cultured in a medium without selection antibiotics for 1-2 passages. If the cells exhibit good condition, you can switch to a medium with selection antibiotics for further passaging. For guidance on selecting appropriate antibiotics, please refer to FAQ12.

Additionally, we recommend adding P/S (Penicillin-Streptomycin) to the culture medium throughout the entire cell culture process to maintain aseptic conditions.

For Adherent Cells (e.g., HEK293):

• Confluence: Avoid over-confluence during culture. If the confluence is too high (exceeding 100%), it may significantly affect cell viability after passaging. Please refer to the passaging methods and precautions in the DS for specific instructions.

• Post-Passaging Issues If cells exhibit poor adherence after passaging due to over-confluence or other reasons, we recommend:

- Removing selection antibiotics from the culture medium.

- Passaging at a higher cell density (e.g., 1×10⁷ cells per T75 flask).

- Resuming normal passaging only after cell viability has recovered.

For Suspension Cells (e.g., Jurkat and Raji):

• Cell Density: Avoid excessively high cell density. If the density is too high (exceeding 3×10⁶cells/mL), it may significantly affect cell viability after passaging. Please refer to the passaging methods and precautions in the DS for specific instructions.

• Post-Passaging Issues: If cells exhibit poor viability after passaging due to high density or other reasons, we recommend:

- Removing selection antibiotics from the culture medium.

- Passaging at a lower density (e.g., 1×10⁵-2×10⁵ cells/mL).

- Resuming normal passaging only after cell viability has recovered.

Always monitor cell health and adjust protocols as needed to maintain optimal growth conditions.

We recommend starting with a transparent 96-well plate for cell seeding (refer to the specific experimental protocol for details). This allows for easy observation of cell status and determination of whether the cell density is appropriate. Once the experimental conditions are optimized, you can transition to other suitable culture plates.

For recommended cell seeding densities, please refer to FAQ9.

For a list of commonly used culture plates and consumables, see the product experimental protocol.

No, it is not necessary to add selection antibiotics.

For 96-well plates, we recommend seeding cells such that they reach approximately 80% confluence after overnight culture before conducting functional experiments. You can start by following the cell seeding density recommended in the ACRO experimental protocol or by testing a gradient of different cell densities to determine the optimal conditions for your experimental system.

During the initial stages of functional experiments, you can start by testing the protein or drug concentrations recommended by ACRO. However, due to variations in reagents or experimental conditions, we recommend conducting preliminary optimization experiments to determine the optimal concentration best suited for your specific experimental system.

We recommend prioritizing the use of culture media and serum from the manufacturers specified in the DS. However, you may also choose to use comparable alternatives or other suitable culture media and serum (e.g., Gibco) for testing and cultivation.

For selection antibiotics, we highly recommend using the brands specified in the DS. The activity of antibiotics may vary between manufacturers, so if you choose to use a different brand, it is essential to validate whether the concentration recommended in the ACRO culture protocol is suitable.

Regardless of the brand used, we recommend maintaining a backup culture without selection antibiotics to avoid potential cell loss due to inappropriate antibiotic concentration.

We highly recommend using the protein reagents from the manufacturers specified in the experimental protocol, as their activity has been validated by us. If you choose to use protein products from other manufacturers, we recommend conducting a concentration optimization based on the recommended concentrations in the protocol to identify the appropriate concentration for your experiments.

For both adherent and suspension cells, we recommend using 90% FBS + 10% DMSO (V/V) as the freezing medium. Alternatively, you may choose commercial cell freezing media or other suitable freezing media commonly used in your laboratory.

Recommended freezing density: 5×10⁶ - 1×10⁷cells/mL.