1.Prepare cell suspension of the required concentration, then mix it with medium evenly in a container. A volume of 30-50ml per layer is recommended .

2.Slowly add the mixed solution into the 5-layer Cell Culture Flask with a serological pipette. To avoid foam orbubbles, it is recommended to set the pipette firmly against the wall, enable the stream to flow along the slope, and save a little liquid in the pipette each time.

Notes: While a 10ml pipette can disperse the medium at the bottom, a 25ml pipette will only reach up to the NEST mark to disperse the medium.

3.Position the Multi-layer Flask upright with the NEST markfacing you, tilt it 45° clockwise and let stand in this position for a while to level the liquid in each layer.

4.Gently lay it flat onto the workbench with NEST markfacing upwards.

5.Gently shake it from side to side to distribute cells evenly onto culture surfaces.

Notes: be careful to shake gently while avoid foam or bubbles and spilling liquid from each layer.

6.Transfer the flask to the incubator for incubation

Culture medium removal

1.Aspiration

Tilt the flask 45° clockwise with the NEST mark facing you. counter-clock wise to a 45° angle while inverting the Multi-Flask toward you. Then reach the serological pipette into the bottom for fully aspiration.

2.Pouring

Tilt the flask 45° counterclockwise with the NEST markfacing you, pour the spentmedia from the flask.

Tips: A NEST 10mL serological pipette is suggested for fully aspiration.

Cell harvesting

1.Rinse off the residual serum with buffer, add digestion solution (≥5mL per layer)and mix evenly. Then, follow Steps 3-4 to distribute to dissociating reagent to each layer.

2.Let stand for 2 min, then neutralize and mix with inactivating solution following steps 3-4. Gently swirl to dislodge cells completely.

3.Transfer the solution in a centrifuge tube or other containers by aspiration or pouring.

4.Rinse the flask with buffer for three times, then transfer the buffer into the centrifuge tube for passage and counting.

Tips: Search "NEST Multi-layer Flask" video on YouTube(@nestwuxi4075).