The Cell Must Be Checked. Have You Ever Done It?

This Paper Describes A Typical Workflow And The Necessary Steps For Cell Operation. In Order To Successfully Apply Mammalian Cell Lines, They Must Grow Under Controlled Conditions And Need Their Own Specific Growth Medium. In Addition, In Order To Ensure Consistency, Their Growth Must Be Monitored Regularly. When The Cell Line Reaches About 80% Confluence, It Must Be Subcultured To Ensure The Normal Growth And Health Of Cells. 80% Coincidence Means That 80% Of The Surface Of The Culture Container Is Covered By Cells& Nbsp; 1.  Why Do I Need To Pass On My Cells? Once Cell Culture Starts, It Cannot Grow Indefinitely Due To The Increase In The Number Of Cells, The Consumption Of Nutrients And The Increase Of Toxic Metabolites, Which Eventually Leads To Cell Death. In Addition, Researchers Usually Conduct Multiple Experiments On Cells, So They Do Not Want To Use Up All Cells At Once. For Passage Or Division Of Cells, By Removing The Culture Medium And Transferring Cells To The Fresh Growth Medium, The Cells Are Given Fresh Nutrition And The Toxic Metabolites Are Also Removed, And The Cells Will Be Given New Vitality To Continue To Grow& Nbsp; 2.  Cell Growth Curve: The Growth Curve Of Cells In The Culture Medium Consists Of Four Stages: The Incubation Period (lag Period) Before The Start Of Growth, The Exponential Growth Period (logarithmic Period), The Stable Period When The Number Of Cells Increases Rapidly And Slows Down Gradually, And The Death Period When Cells Die, Because Of Lack Of Nutrition And Wrong Living Conditions. In The Logarithmic Phase, The Medium Should Be Changed, And Cells Should Be Subcultured Before Reaching The Fixed Phase, As Shown In Figure 1; Fig. 1 Please Note That In Order To Maintain The Healthy And Active Growth Of Cells, It Is Necessary To Update The Growth Medium And Pass It On Regularly. According To The Different Cell Types, The Medium Can Be Changed Several Times In The Logarithmic Phase. The Best Time For Passage Of Cells Is Between The Logarithmic Phase And The Fixed Phase, That Is, Before The Cells Reach Confluence& Nbsp; 1. Why Are Cells Passaged With Trypsin? The Most Common Way To Prepare Cells For Subculture Is To Use Proteolytic Enzymes Such As Trypsin To Destroy The Connection Between Cells And Substrate. The Combination Of Trypsin And Ethylenediamine Tetraacetic Acid (EDTA) Will Cause Cells To Detach From The Growth Surface. Trypsin Cut Off The Local Adhesion Of Cells Fixed On The Culture Dish, And EDTA Was Used As Calcium Chelating Agent& Nbsp;& Nbsp;& Nbsp; By Removing Calcium, Cadherin, Which Participates In The Interaction Between Cells, Is Destroyed And Cells Separate From Each Other. Once Separated From The Growth Surface And Surrounding Cells, It Can Be Easily Separated And Grown In A New Cell Culture Dish& Nbsp;& Nbsp; Cell Culture Conditions And Subculture Methods Vary Depending On Cell Type. Figure 2 Describes The Basic Steps In The Cell Culture Process. In The Whole Cultivation Process, It Is Very Important To Work In A Pollution-free Environment. Cells Were Examined At The Beginning, Trypsinization, Cell Count, And After Division. In Order To Achieve Consistent Results, It Is Also Important To Maintain Good Records And Documentation& Nbsp;& Nbsp; 2.4 Steps To Determine Whether Cells Can Be Passaged: 1; Take The Cells Out Of The Incubator And Place Them Under The Microscope For Rapid Cell Examination. Cells Should Be Examined Under A Microscope Every Day To Ensure That They Are Healthy (no Pollution, Few Dead Cells) And Grow As Expected. 2.  The Cells Should Be Mainly Attached To The Bottom Of The Culture Dish Or Flask, And The Culture Medium Should Be Pink Orange. Phenol Red, The PH Indicator, Turns Yellow When Acidified. At This Time, It Should Be Noted That There Are Two Situations: One Is That The Cell Density Is Too High, And It Has Reached The Plateau Of Growth; The Other Is That There Are Pollutants In The Cell. 3.  It Is Very Important To Take Photos With A Microscope And Record The Cell Status To Ensure The Consistency Of The Experimental Results& Nbsp; 3. Pass On My Cells 1. The Pipette Transfers The Culture Medium From The Cell Dish To The Waste Container. The Cells Were Carefully Washed With 5ml Of Pre Heated PBS Without Calcium And Magnesium For Up To Three Times To Remove Fetal Bovine Serum (FBS) From The Residual Medium. Fetal Bovine Serum Inhibits Trypsin. 2. Add Pre Heated Trypsin/EDTA Of Corresponding Specifications;, For Example: T25 Bottle 1-2ml Or So, Gently Rotate To Cover All Cells At The Bottom Of The Culture Dish. 3.  The Cells Were Incubated At 37 ° C For 2-3 Minutes Or At Room Temperature For 3-5 Minutes To Separate Them. Different Cell Lines Need Different Incubation Time Of Trypsin. In Order To Avoid Damage To Cells By Excessive Trypsin, The Cells Were Examined Under A Microscope Every 2 Minutes. After The Cells Become Elliptical And Transparent, When The Edge Of Caco-2/hepG2 Cells Starts To Float, Add More Than Double The Complete Medium To Neutralize, Transfer The Cell Suspension To A 15ml Test Tube, And Rotate At 1000 Rpm For 5min. 4.  Suck The Supernatant, Add 2 Ml Of Complete Culture Medium To Re Suspend The Cells, And Then Separate Or Freeze Them& Nbsp;& Nbsp; 4.  Cell Count: 1. Mix 100 µ L Cell Suspension With An Equal Amount Of 0.4% Trypan Blue Solution. Trypan Blue Can Selectively Penetrate The Cell Membrane Of Dead Cells And Dye It Blue, But It Will Not Be Absorbed By Living Cells. Place The Cap On The Counting Surface And Prepare The Hematometer. 2.  Place The Tip Of The Pipette At The Edge Of The Cover Glass, Load The Cell Suspension Into The Counting Chamber (each Counting Area Is About 4 µ L), And Gently Discharge The Cell Suspension. The Area Under The Caprock Is Silted By Capillarity. In Most Cases, The Chamber Has Two Counting Areas That Can Be Loaded Independently. 3.  Place The Chamber On The Microscope Table And Focus The Cells. The Square Pattern Of The Counter Grid Varies With The Type Of Cavity. The Fox Rosenthal Counting Room You See Here Has 16 Areas With An Area Of One Square Millimeter, Each Area Is Surrounded By Three Lines. Each Square Is Further Divided Into 16 Smaller Squares. Calculate All The Cells In A 16 Square Area, As Shown In The Figure. To Avoid Calculating Cells Twice At The Edge Computing Of The Area, Only Those Cells On The Straight Lines On Both Sides Of The Square Are Calculated. In This Example, You Should Count The Cells That Touch The Upper Left Limit (the Thicker Red Line). The Lower Limit And Right Limit Of Cell Touch Should Not Be Taken Into Account. Count The Living Cells And Dead Cells According To Five Rectangles Of One Square Millimeter. To Calculate, You Need To Combine The Counting Results Of All 5 Squares. In Order To Improve The Measurement Accuracy, The Additional Square Of The Counting Chamber Can Also Be Calculated& Nbsp; The Calculation Formula Of Cell Concentration Is As Follows: What Problems Will You Encounter When Operating Cells? The WeChat Background Will Leave Us A Message, And The Technology Will Count The Answer In Future Official Account Articles& Nbsp;