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Nine Basic Techniques Of Mammalian Cell Culture

source:Qida organism  views:1604  time:2021-06-11

1. Purified Air Before Any Tissue Culture, Open The Fresh Air System For At Least 15 Minutes To Ensure That There Is Clean Air Flowing In. Always Make Sure Nothing Covers It. Turn On The UV Lamp 3-4 Times A Week For A Maximum Of Half An Hour, Which Can Help Keep Your Laboratory Free Of Pollution, But You Should Remember That UV Will Only Kill The Microorganisms Directly Exposed To It. Ultraviolet Radiation Is Also Harmful To Eyes And Skin, So Please Keep The Light Off When Using The Light Shield. Wipe The Super Clean Table With Disinfectant Before And After Work (most People Use 70% Ethanol, But You Can Also Use Incidin Wipes). 2. Keep Away From Pollutants Although You May Be The Main Pollution Source Of The Experiment, You Must Take All The Precautions You Can To Prevent Cells From Being Polluted. Wipe Everything In The Lab With 70% Ethanol, Including Your Gloved Hand! You Should Also Take Down All The Jewelry And Watches You Wear Before Starting. In Addition, Avoid Unnecessary Conversation, Because Conversation Will Produce Microbial Aerosol, Which May Enter The Super Clean Platform. Planning Your Experiment Thoroughly Before Entering The Laboratory Is Another Good Way To Prevent Pollution. This Sounds Trivial, But It Often Solves Most Problems Before They Occur. With Proper Planning, You Can Know Exactly What Materials You Need To Bring Into The Laboratory, And You Can Clean Them Before Bringing Them In. 3. Separator! Keep The Laboratory Clean And Tidy. It Is Better Not To Wear A Cover For The Lockers. Chaos Not Only Makes It Difficult To Clean Your Work Area, But Also Destroys The Laminar Airflow Around The Work Area. Ensure That Only Required Materials Are Brought Into The Work Area. This Will Give You Enough Space To Move Freely Without Causing Leakage. In Addition, When The Liquid Container Cannot Be Used Immediately, Please Tightly Cover It. Prevent Leakage, But If Any Leakage Occurs, Wipe It With 70% Ethanol Immediately. Finally, The Pipette Should Not Be Directly Inserted Into The Culture Medium Bottle, But The Solution Should Be Poured Into The Disposable Sterile Tube First, Which Can Not Only Facilitate Management But Also Avoid Large-scale Pollution. 4. Make Sure That The Cells Do Not Leave The Incubator For Too Long. When You Prepare Something, Make Sure That Your Cells Do Not Stay Outside For Too Long. The Comfortable Area Of The Incubator Is Usually About 37 ° C And 5% Carbon Dioxide. Sudden Changes In Temperature (especially When The Air Conditioner Is Turned On) Will Cause Cells To Become Restless, So They Are Put In A Sterile Incubator. This Also Means That Any Culture Medium Or Washing Buffer In Contact With Cells, Such As PBS (phosphate Buffered Saline), Should Be At 37 ° C. This Is Also The Purpose Of Most Laboratories To Have A 37 ° Water Bath. 5. Pay Attention To The Shape And Size Of Cells. You Should Put A Light Microscope Near The Ultra Clean Table, So That You Can Observe Your Cells Regularly (before Entering The Laboratory And When Putting Them Into The Incubator). This Will Also Help You Become Familiar With The Morphology Of The Cell Line You Are Using In Order To Identify Cross Contamination. Even Though Some Mammalian Cell Lines Have Similar Morphology, Microscope Is Necessary To Observe Whether Cells Are Polluted. 6. Reasonably Set Up Cell Passage. Don't Be Lazy To Divide Cells, But Form A Routine. For Most Rapidly Growing Cell Lines (such As HEK293, Which Proliferates Every 16 Hours), Twice A Week Is Usually Effective. For Example, If You Dilute Cells 1:5 On Monday For Passage, You Should Be Able To Divide The Next Thursday Or Friday At The Latest. It Is Best To Divide Cells Before 80-85% Of The Cell Plates Converge, Because Cells Are At Their Peak Of Growth At This Time. If They Reach The Junction Point, They Will Enter The Flattop Period, And They Will Stop Growing (contact Inhibition). After Passage, They Will Take Time To Recover. This May Change Their Growth Rate And Morphology, Making Them Unsuitable For Experiments That Require Reproducible Results. Most Cell Cultures Contain The PH Indicator Phenol Red (pH Range 6.0 (yellow) - 8.0 (red)). The Blood PH Value Is Usually Around 7.35-7.4, So Mammalian Cells Are Very Sensitive To The PH Value. The Color Between Orange And Light Orange Usually Indicates That Cells Need To Change Fluid Or Have Enough Growth. By Secreting Lactic Acid (a By-product Of Cell Metabolism, Which Is Toxic To Cells), The Surrounding Medium Becomes Acidic. At This Time, If The Cell Density Is Too High, The Cells Will Choose To Pass On. 7. Cell State Is Power& Nbsp; When A New Cell Line Is Used To Start Culture, The Cells Will Grow, Divide Them Into Several Equal Vials, Freeze Them, And Store All But One In Liquid Nitrogen. The Frozen Vials Form Your Main Inventory, And You Can Use The Remaining Vials To Grow And Freeze Additional Cells, Which Can Serve As Your Work Inventory. This Ensures That You Have Many Cells Stored In Their Original State Once The Working Cells Become Too Old Or Contaminated. You Still Have The Previous Stock Cells To Replace. This Also Ensures That Anyone Who Needs New Cells In The Laboratory Can Bring A Bottle Of "original State" Master Stock, Rather Than "borrowing" Cells That Have Been Passed Down Many Times And May Be Contaminated& Nbsp; 8. Pay Attention To The Cell Passage Times Used In The Experiment: Pay Attention To The Passage Times. Cells Will Change Their Growth Rate And Morphology When They Have A High Transmission Number (> 30-40 Times), Depending On The Cell Line, So Old Cells Should Be Retired! On The Contrary, The Young Cells Of 3-4 Generations Are Too Young To Be Used In Important Transfection Experiments Immediately. Additional Passages Can Allow Your Cells To Grow At A Steady Rate& Nbsp; It Is Good Practice To Stick Your Name, Date, Cell Line You Use And Its Channel Number On Your Plate And/or Flask. Especially In A Laboratory Where Many People Are Engaged In Different Work, This Will Help You Avoid Cross Contamination Caused By Accidental Mixing, And Also Help You Keep Good Experimental Records. 9.  Autoclave Is A Carnival Of Cells! Last But Not Least, Any Laboratory Materials Used For Reuse (e.g. Pasteur Pipettes For Liquid Waste) Should Be Disinfected Before Each Use. In Addition, All Contaminated Liquid And Solid Wastes Should Be Autoclaved Before Treatment! Cultivate Happiness!
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