Accutase Protocol for the Dissociation of Stem Cells

The following protocols offer guidance for using StemPro Accutase reagent, a serum- and animal-component-free reagent optimized for sensitive stem and progenitor cells. Whether you are working with human embryonic stem cells (hESCs), neural stem cells (NSCs), or neurosphere cultures, these workflows offer step-by-step instructions to help achieve high-viability single-cell suspensions with minimal impact on surface markers and pluripotency.

1. Aspirate the medium from culture dish and wash with 4 mL of DPBS without calcium and magnesium.
2. Aspirate DPBS and add 2 mL of Accutase reagent to culture dish.
3. Incubate for 2 to 5 minutes at 37°C until individual single cells start to round up.
4. Gently rinse to remove cells from the plate’s surface.
5. Transfer cell suspension to 15 mL conical tube. Gently pipette up and down until cells are in a single-cell suspension.
6. Add 8 mL of medium to rinse any remaining cells from the dish’s surface and transfer to the conical tube (from Step 5).
7. Take a 20 µL sample of the cell suspension to determine viable cell density.
8. Centrifuge conical tube containing the cell suspension at 200 g for 4 minutes.
9. Aspirate supernatant, resuspend in fresh medium and plate on coated dish(s). Incubate at 37°C in a humidified atmosphere of 5% CO₂ in air.

1. Aspirate the medium from culture dish and wash with 4 mL of DPBS without calcium and magnesium.
2. Aspirate DPBS and add 2 mL of Accutase reagent to culture dish.
3. Incubate for 2 to 5 minutes at 37°C until individual single cells start to round up.
4. Gently rinse to remove cells from the plate’s surface.
5. Transfer cell suspension to 15 mL conical tube. Gently pipette up and down until cells are in a single cell suspension.
6. Add 8 mL of medium to rinse any remaining cells from the dish’s surface and transfer to the conical tube (from Step 5).
7. Take a 20 µL sample of the cell suspension to determine viable cell density.
8. Centrifuge conical tube containing the cell suspension at 200 g for 4 minutes.
9. Aspirate supernatant, resuspend in fresh medium and plate on coated dish(s). Incubate at 37°C in a humidified atmosphere of 5% CO₂ in air.

1. Remove neurosphere cell suspension from culture dish and transfer to a 15 mL conical tube.
2. Let neurospheres settle down in the tube (~2 to 5 minutes) before proceeding to Step 3. Alternatively, the cells can be centrifuged at 100 g for 1 minute.
3. Gently aspirate medium leaving the neurospheres at the bottom of tube with approximately 100 μL of media remaining.
4. Resuspend neurospheres in 5 mL DPBS without calcium and magnesium.
5. Let neurospheres settle down in the tube (~2 to 5 minutes) before proceeding to Step 6. Alternatively, the cells can be centrifuged at 100 g for 1 minute.
6. Gently aspirate DPBS leaving the neurospheres at the bottom of tube with approximately 100 μL of DPBS remaining.
7. Add 1 mL of Accutase reagent to the neurospheres and incubate 10 minutes at room temperature.
8. Using the proper sized pipette tip (i.e., 1,000 μL), pipette up and down until all the neurospheres are in a single cell suspension.
9. Add 4 mL of fresh medium to the tube.
10. Centrifuge the cells at 200 g for 4 minutes.
11. Gently aspirate the supernatant.
12. Resuspend cells in fresh medium, transfer to a new culture dish and incubate at 37°C in a humidified atmosphere of 5% CO₂ in air.

How long does Accutase reagent take to work?

Accutase reagent typically detaches adherent cells within 1–10 minutes when incubated at 37 °C. For hESCs grown on Geltrex matrix, the recommended incubation is 2–5 minutes at 37 °C until cells begin to round up.

No, Accutase reagent does not require a separate neutralization step with serum or other inhibitor. The reaction can be stopped simply by dilution with DPBS or culture medium.

The exact incubation time of Accutase reagent depends on cell type and coating, but the following is recommended:

• For general adherent cells: 1–10 minutes at 37°C.
• For hESCs on Geltrex matrix: 2–5 minutes at 37°C until individual cells round up.
• For neurospheres: up to 10 minutes at 37°C (with gentle pipetting) in some protocols.

Always monitor microscopically and adjust time to avoid over-dissociation of cells.

Accutase reagent offers multiple advantages over traditional trypsin:

• Maintains higher viability of stem cells and primary cells compared to animal-origin enzymes like trypsin.
• More gentle dissociation, helping preserve cell surface proteins and epitopes, which is important for downstream applications such as flow cytometry, marker analysis, and maintaining pluripotency.
• It is animal-component-free and serum-free, reducing variability and risk of contamination from mammalian- or bacterial-derived components.
• Designed for sensitive cells including hESCs, hiPSCs, MSCs, NSCs, and primary cells—where trypsin can cause damage or loss of surface markers.