A few cell lines that Accutase™ has been shown to detach without harm:

Accutase performs exceptionally well in detaching cells for:

Accutase is formulated at a concentration that is ready to use, once defrosted. (Note: Never defrost a bottle of Accutase at 37°C.) A defrosted bottle of Accutase can be removed from the refrigerator and immediately applied to cells. It does not need to be and should not be pre-warmed to 37°C. Accutase contains proteolytic and collagenolytic enzymes to gently break down the cell adhesion structure on the outside of cells that attaches them to the bottom of the flask.

This entire procedure should be done in a laminar flow hood using proper aseptic technique.

1. Carefully aspirate all of the media from the cell culture flask. (Rinsing with PBS is not necessary.)
2. Immediately add enough Accutase to the flask to cover the cells. (Typically 2.5 to 5ml for a T25 flask depending upon confluency and density of the cell culture.)
3. Set the flask aside at room temperature (RT) for 5 to 10 minutes up to a maximum of 1 hr. Check the flask frequently to see if the cells have rounded rather than merely shrunken or no longer appear "spidery" while remaining attached to the bottom of the flask.
4. Once the cells have turned into "balls", smack the flask against the palm of your hand to dislodge any "stickers".
5. Gently disperse the cells and take a sample of the cell suspension to determine the viable cell density.
6. Add an aliquot of the detached cells to fresh media in new flasks. Place the flasks into the 37°C incubator. No neutralization steps are required. The cells will reattach within a few minutes depending upon cell type.

Primary macrophage cultures may be derived from density gradient separated whole blood or bone marrow by culturing these isolates in the presence of specific growth factors in order to encourage the growth, expansion and differentiation of the macrophage cell type. A literature search will yield several in-depth protocols which out-line these isolation techniques in detail. Once harvested and put into culture at 37^oC in a 5% CO2 incubator the macrophage progenitor cells will adhere to tissue culture flasks and will not be washed away when the media is changed in order to remove any non-adherent cells and allow for macrophage differentiation/maturation.

To remove the Primary macrophage cultured cells from the tissue culture plates for further analysis:

1. Remove the supernatant from the flask (T25) or tissue culture dish (15mm).
2. Wash the plate or dish with 5ml sterile PBS and remove it.
3. Add 5ml of Accutase™ to the flask or dish and incubate at room temperature for 10-15 minutes. Inspect the flask under the inverted microscope to look for cell “shrinkage” or detachment.
4. Gently swirl the flask and add another 5ml of Accutase™. Incubate the cells another 10-15 minutes at room temperature. Re-suspend the cells by trituration (pipetting up and down several times) being careful not to cause bubbles.
5. Count the cells and adjust the concentration to be used for further analysis.
   NOTE: This procedure may also be carried out on ice which may decrease the incubation times needed.

Passaging Macrophage Cell Lines such as DH82 and RAW264.7
Using Accutase™ Cell Detachment Solution

For those investigators who prefer to utilize immortalized cell lines rather than isolate primary cultures, Accutase™ may be used to passage their macrophage lines in a similar manner to the techniques used for most other adherent cell lines.

1. Remove flask (T25) from the incubator and examine it under the inverted microscope to check for cell confluency.
2. Transfer the flask to the tissue culture hood for cell passaging. Remove the media from the flask. Wash the flask 2 times with 5ml of sterile PBS; fully remove after each addition.
3. Add 5ml of Accutase™ to the flask and incubate it at room temperature for 5-10 minutes.
4. Inspect under the microscope for classic signs of cell detachment ie, “shrinkage” or “rounding”.
   a. If there is significant signs of “rounding”, then transfer the plate back to the hood and triturate (pipette up and down several times) being careful to not create bubbles.
   b. If cells do not appear significantly “rounded” then allow them to incubate and additional 2-3minutes and proceed as in step “a” above.
5. Passage cells at a 1:2 or 1:4 ratio every 3-6 days.

Please note that different adherent cells stick to tissue culture plastic with varying degrees of adherence. For this reason the incubation time required for detachment can vary with some cell types needing more or less time to detach. In the case of extremely tenaciously adherent cells our stronger formulation, Accumax may work better.

Dissociation of adherent human or rat NSCs

1. Aspirate the medium from culture dish.
2. Add 2 mL of Accutase™ 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 off of 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 off of the dish’s surface and transfer to the conical tube (from Step 5).
7. Take a 20uL sample of the cell suspension to determine viable cell density.
8. Centrifuge conical tube containing the cell suspension at 200g for 4 minutes.
9. Aspirate supernatant, resuspend in fresh medium and plate on coated dish(s).  Incubate at 36 to 38°C in a humidified atmosphere of 4 to 6% CO2 in air.

Dissociation of human or rat neurosphere cultures

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 100g 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.
5. Let neurospheres settle down in the tube (~2 to 5 minutes) before proceeding to Step 6. Alternatively, the cells can be centrifuged at 100g for 1 minute.
6. Gently aspirate DPBS leaving the neurospheres at the bottom of tube with approximately 100 μL of DPBS remaining.
7. Add 1mL of Accutase™ to the neurospheres and incubate 10 minutes at room temperature.
8. Using the proper sized pipette tip (i.e.1000 μl), pipette up and down until all the neurospheres are in a single cell suspension.
9. Add 4mL of fresh medium to the tube.
10. Centrifuge the cells at 200g for 4 minutes.
11. Gently aspirate the supernatant.
12. Resuspend cells in fresh medium, transfer to a new culture dish and incubate at 36 to 38°C in a humidified atmosphere of 4 to 6% CO2 in air.

1. Yotsumoto, Karen, et al. "Amelogenin Downregulates Interferon Gamma-Induced Major Histocompatibility Complex Class II Expression Through Suppression of Euchromatin Formation in the Class II Transactivator Promoter IV Region in Macrophages." Frontiers in Immunology 11 (2020): 709.
2. Inamori, Sachiko, et al. "Modeling early stages of endoderm development in epiblast stem cell aggregates with supply of extracellular matrices." Development, Growth & Differentiation (2020).
3. Deng, Xiaoyue, et al. "Characterization of human induced pluripotent stem cells carrying homozygous RB1 gene deletion." Genes to Cells (2020).
4. Kikuchi, Tetsutaro, and Tatsuya Shimizu. "Thickness-wise growth technique for human articular chondrocytes to fabricate three-dimensional cartilage grafts." Regenerative therapy 14 (2020): 119-127.
5. Tozaki-Saitoh, Hidetoshi, et al. "Involvement of exchange protein directly activated by cAMP and tumor progression locus 2 in IL-1β production in microglial cells following activation of β-adrenergic receptors." Journal of Pharmacological Sciences (2020).
6. Kusumoto, Junya, et al. "OPN4 belongs to the photosensitive system of the human skin." Genes to Cells 25.3 (2020): 215-225.
7. Shigeto, Kawai, et al. "Three-dimensional culture models mimic colon cancer heterogeneity induced by different microenvironments." Scientific Reports (Nature Publisher Group) 10.1 (2020).

• Typical Cell Passaging Instructions using Accutase™
• Macrophage Passaging Protocol
• Dissociation of NSCs & Neurospheres
• Defrosting Accutase™ and Accumax™ Correctly