Cell culture guidelines Abcam（细胞培养的指导方针Abcam）.pdf

Cell culture guidelines The following is a general guideline for culturing of cell lines. All cell culture must be undertaken in microbiological safety cabinet using aseptic technique to ensure sterility. 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) (b) Glass 9” Pasteur pipettes (c) 70% ethanol. Be sure to spray all surface areas 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. Preparation of cell growth medium Before starting work check the information given with the cell line to identify what media type, additives and recommendations should be used. 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). Check which culture media and culture supplements the cell line you are using requires before starting cultures. Culture media and supplements should always be sterile. Purchase sterile reagents when possible, only use under aseptic conditions in a culture hood to ensure they remain sterile. General example using DMEM media: DMEM - Remove 50 ml from 500 ml bottle then add the other constituents. 450 ml 10% FBS 50 ml 2 mM glutamine 5 ml 100 U penicillin / 0.1 mg/ml streptomycin 5