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General details of cell culturing

互联网2013-11-13

916

实验试剂

General example using DMEM media
1. 450 ml DMEM - Remove 50 ml from 500 ml bottle then add the other constituents.
2. 50 ml 10% FBS
3. 5 ml 2 mM glutamine
4. 5 ml 100 U penicillin / 0.1 mg/ml streptomycin

实验步骤

1. Preparing an aseptic environment

    1) Hood regulations
        a. Close hood sash to proper position to maintain laminar air flow
        b. Avoid cluttering

2) Autoclaving
a. Pipette tips (or can be purchases pre-autoclaved, DNAse/RNAse free)

3) b. Glass 9” Pasteur pipettes

4) c. 70% ethanol (Be sure to spray all surface areas)

2. Preparation of cell growth medium

1) Before starting work check the information given with the cell line to identify what media type, additives and recommendations should be used.

2) Most cell lines can be grown using DMEM culture media or RPMI culture media with 10% Foetal Bovine Serum (FBS), 2 mM glutamine and antibiotics can be added if required (see table below).

3. Creating the correct culturing environment

Most cell lines will grow on culture flasks without the need for special matrixes etc. However, some cells, particularly primary cells, will require growth on special matrixes such as collagen to promote cell attachment, differentiation or cell growth. We recommend reviewing the relevant literature for further information on the cells you are culturing.

The following is an example for endothelial and epithelial cells:

For human cells, coat flasks with 1% gelatin. Alternatively, for other cell types such as BAEC, flasks can be coated with 1% fibronectin.

1) Prepare 10mL of coating solution composed of 1% gelatin or 1% fibronectin by diluting with distilled water, followed by filtration. This is efficient to coat about 5 flasks.

2) Pipette coating solution into flask. Rock back and forth to evenly distribute the bottom of the flask. Let sit in an incubator for 15-30 minutes.

3) Completely remove coating solution by aspirating before seeding.

4. Checking cells

1) Cells should be checked microscopically daily to ensure they are healthy and growing as expected.Attached cells should be mainly attached to the bottom of the flask, round and plump or elongated in shape and refracting light around their membrane.Suspension cells should look round and plump and refracting light around their membrane. Some suspension cells may clump.Media should be pinky-orange in colour.

2) Discard cells if:

They are detaching in large numbers (attached lines) and/or look shrivelled and grainy/dark in color.

They are in quiesence (do not appear to be growing at all).

注意事项

1. All media, supplement and reagents must be sterile to prevent microbial growth in the cell culture. Some reagents and supplements will require filter sterilization if they are not provided sterile.

2. Check which culture media and culture supplements the cell line you are using requires before starting cultures. Culture media and supplements should be sterile. Purchase sterile reagents when possible, only use unders aseptic conditions in a culture hood to ensure they remain sterile.

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