User guide for seeding cells into a 5-layer Cell Culture Flask
1.Prepare cell suspension of the required concentration, thenmix itwith mediumevenly in a container. A volume of 30-50ml per layerisrecommended .
2.Slowlyadd the mixed solutioninto 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 thestream to flow along the slope, and save a little liquid in the pipette each time.
Notes: Whilea 10mlpipette candispersethe mediumat the bottom, a25ml pipette will only reach up to the NEST mark to disperse the medium.
3.Positionthe Multi-layer Flask upright with the NEST markfacing you,tilt it 45° clockwise and let stand in this position for a while to level theliquid in each layer.
4.Gently lay it flat onto the workbench with NEST markfacing upwards.
5.Gently shake itfromsidetoside to distribute cells evenly onto culture surfaces.
Notes: be careful toshake gently toavoid foamor bubbles and spilling liquid from each layer.
6.Transferthe flaskto the incubatorfor incubation
Culture mediumremoval
1.Aspiration
Tilt the flask45° clockwise with the NEST mark facing you. counter-clock wise to a 45° angle while inverting the Multi-Flask toward you. Thenreach the serological pipette into the bottomfor fully aspiration.
2.Pouring
Tilt the flask45° counterclockwise with the NEST markfacing you, pour the spentmedia from the flask.
Tips: ANEST 10mL serological pipetteis suggested for fully aspiration.
Cellharvesting
1.Rinse off the residual serumwith 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).
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