Material:
high-density polyethylene cap
polystyrene flask
Quality Level: 200
Sterility: sterile;
γ-irradiated
Feature: 5
layer
Packaging: pack
of 8
manufacturer/tradename:
Millicell®
parameter: 45
°C max. temp.
technique(s): cell
culture | mammalian: suitable
H:
51 mm , excluding cap
74 mm , including cap
L: 22.2 cm
W: 12 cm
surface area: 1000 cm2
working volume: 200-300 mL
binding type: Tissue
Culture (TC)-treated surface
shipped in: ambient
Preparation Note
- To
seed cells, fill the Millicell HY T-1000 Flask with the desired growth
media.
- Add
desired number of cells to achieve typical seeding density.
- Mix
the cells and media by raising the end of the flask and pipetting the
solution up and down several times until a homogeneous suspension is
achieved. Note: Failure to adequately mix cells into a homogeneous
suspension may result in an uneven distribution of cells across the layers
of the Millicell HY Flask.
- Briefly
lay the flask on its side to equilibrate the liquid volume equally among
the layers of the flask.
- Tilt
the flask on its end to partition the liquid volume for each layer of the
flask.
- Lay
the flask down to spread the cell suspension evenly across the entire
surface of each layer.
- Depending
on the volume it may be necessary to rock the flask on all four corners to
ensure that the entire surface wets. Note: If the liquid volume on any of
the layers changes during the wetting procedure (e.g., spilling of liquid
from top layer to lower layers), repeat steps 4–6 to ensure even
distribution of liquid among all layers.
- Return
the flask to a vertical position when transporting to the incubator. Lay
the flask flat in the incubator (as in step 6) and culture cells under
appropriate conditions.
- If
media exchange is required, either aspirate or pour the media
from the Millicell HY T-1000 Flask. Note: Due to the larger surface area
of the Millicell Flasks, it may be necessary to let the flask stand on its
side for several minutes following each aspiration so that all residual
liquid pools to a common point, allowing for a second aspiration to
completely remove the remaining liquid.
- Add
appropriate volume of fresh media, and repeat steps 4–8.
- To
harvest cells, remove media as in step 9.
- Add
desired amount of appropriate wash solution (e.g., PBS or 0.02% EDTA) to
the flask and repeat steps 4–7.
- Remove
wash solution as in step 9.
- Add
desired amount of dissociation enzyme (e.g., 0.25% trypsin/EDTA or
equivalent) according to preferred protocol, repeat steps 4–8, and
incubate. Volume of dissociation enzyme required per cm2 does
not need to be altered from T-flask protocol. Note: Ensure that all cells
have completely dissociated from the surface of the flask via observation
under microscope. Failure to allow complete dissociation of cells from
culture surface (such that all cells are freely floating) may result in
reduced cell yields.
- If
using trypsin, add desired volume of inactivating solution, such as
serum-containing media.
- Collect
cells either by aspirating or pouring, as in step 9.
Legal Information
Millicell is a registered trademark of Merck KGaA,
Darmstadt, Germany
