Cell Culture Is No Trivial Matter (1)

  & Nbsp; A Small Cell May Make People Unable To Eat, Sleep, And Be In Dire Straits??? Believe It Or Not, The Whole Experiment Is Planned To Last About Three Months, But Sometimes The First Step Is Blocked, Why? Cells Are Always Poorly Nourished; Oh, My God, It Will Be Three Months Soon, SO Sad, Let's Take A Look At The Main Cell Culture Steps& Nbsp; Choosing The Right Medium Is Important: & Nbsp; & Nbsp; There Is No Fixed Culture Condition For Cultivating A Certain Type Of Cells. Cells Cultured In MEM Are Also Likely To Grow Easily In DMEM Or M199. In A Word, MEM Is The First Choice For Adhesive Cell Culture And RPMI-1640 Is The First Choice For Suspension Cell Culture, And AIM V (12005) Medium (SFM) Is The Best Choice For Serum-free Culture For Various Purposes& Nbsp; What Is GlutaMAX-I? How Do Cultured Cells Use GlutaMAX-I? How Stable Is This Dipeptide& Nbsp; & Nbsp; & Nbsp; GlutaMAX-I  Dipeptide Is A Derivative Of L-glutamine, And Its Unstable Alpha Amino Is Protected By L-alanine. A Peptidase Gradually Cleaves Dipeptides And Releases L-glutamine For Utilization. GlutaMAX-I Dipeptide Is Very Stable. Even After 121 Lb Sterilization For 20 Minutes, GlutaMAX-I Dipeptide Solution Has Minimal Degradation. If L-glutamine Is Almost Completely Degraded Under The Same Conditions& Nbsp; & Nbsp; Phenol Red Is Used As The Indicator Of PH Value In The Medium: Red In Neutral, Yellow In Acid, Purple In Alkaline. Studies Have Shown That Phenol Red Can Mimic The Effects Of Steroid Hormones (especially Estrogen). In Order To Avoid Steroid Reaction, Culture Cells, Especially Mammalian Cells, With Medium Without Phenol Red. Because Phenol Red Interferes With The Detection, Some Researchers Do Not Use The Medium With Phenol Red When Doing Flow Cytometry& Nbsp; What Is The Role Of Sodium Pyruvate In The Medium& Nbsp; & Nbsp; & Nbsp; Sodium Pyruvate Can Be Used As An Alternative Carbon Source In Cell Culture. Although Cells Prefer To Use Glucose As The Carbon Source, If There Is No Glucose, Cells Can Also Metabolize Sodium Pyruvate& Nbsp;& Nbsp; & Nbsp; & Nbsp; Once You Add Serum And Antibiotics To The Fresh Medium, You Should Use It Within Two To Three Weeks. Because Some Antibiotics And Basic Components In Serum Begin To Degrade After Thawing& Nbsp;& Nbsp; & Nbsp; & Nbsp; Most Additives And Reagents Can Be Frozen And Thawed For 3 Times At Most. If The Times Are More, They Will Cause A Certain Level Of Degradation And Precipitation In The Solution Containing Protein, Which Will Affect Its Performance. This Is Especially Evident In The Serum. Some Serum Deposits Are Too Much. The Customer Service Will Tell You That This Is The Serum Protein, And It Is Also The Principle That You Should Not Use It Directly After Centrifugation, Because Your Serum Has No Nutritional Protein After Centrifugation& Nbsp; & Nbsp; & Nbsp; Use The Liquid Trypsin Solution Stored In A 4 ℃ Refrigerator Within One Week Of Dissolution. Trypsin May Start To Degrade At 4 ℃. If It Is Left At Room Temperature For More Than 30 Minutes, It Will Become Unstable & Nbsp; & Nbsp; & Nbsp; At The Same Time, The State Of Cells Is Also Very Important. Some Teachers Think That Tumor Cells Can Be Passed On Indefinitely Anyway, So What If I Use The Cells Of 188 Generations Passed On Before To Do Experiments? If The Cells Are Good In Shape, There May Be No Problem, But If The Cells Are Not Good In Shape, We Should Consider Whether To Use This Cell, Generally, The Bad Morphology Of Cells Involves Three Aspects: 1. The Improper Use Of Culture Medium Seriously Changes The Nutritional Environment Of Cells, And Cells Have To Mutate To Grow In Order To Save Themselves; 2. Incorrect Operation Method, Resulting In Cell Pollution, Etc. 3. Too Many Passages, Because Each Passage Will Cause Slight Damage To Cells, Resulting In Continuous Repair Of Cells After Passage. Derivatives Needed By Repaired Cells, Such As Serum And Culture Medium, May Cause Varying Degrees Of Variation To Cells According To Different Recipes And Batches& Nbsp; So Easy:& Nbsp; & Nbsp; The First Time I Saw A Cell, I Didn't Know Where To Start. It Was Too Delicate. What Should I Do? In Fact, We Just Need To Remember One Thing: Aseptic Operation, Aseptic Operation, Aseptic Operation. Say The Important Things Three Times, And Then We Can Work Hard& Nbsp; Step 1 ：  How Should The Freezing Tube Be Thawed? After The Freezing Tube Is Taken Out, It Must Be Immediately Put Into The 37 C Water Tank For Quick Thawing, Gently Shake The Freezing Tube To Melt It All Within 1 Minute, And Pay Attention That The Water Surface Cannot Exceed The Cover Edge Of The Freezing Tube, Otherwise It Is Easy To Cause Pollution. In Addition, When The Freezing Tube Is Taken Out Of The Liquid Nitrogen Barrel For Thawing, Attention Must Be Paid To Safety To Prevent The Bursting Of The Freezing Tube. Note: Centrifuge Is Not Necessary For Resuscitation. Except For A Few Cells That Are Specifically Noted To Be Sensitive To DMSO, Most Cell Lines (including Suspended Cells) Should Be Directly Put Into The Culture Bottle Containing 10-15ml Of Fresh Medium After Thawing, And The Fresh Medium Should Be Replaced The Next Day To Remove DMSO. This Can Avoid The Problem That Most Cells Cannot Grow Or Attach After Thawing& Nbsp; Step 2: How To Cultivate& Nbsp; & Nbsp; & Nbsp; & Nbsp; The Key Points Of Cultivation Are Still In The Process Of Generation. When I Think Of Generation, God, Is It Very Complicated? First Of All, We Should Wait Until The Cells Reach The Density Of 80% - 85% Before Operating. Why Is This Density? Because This Density Is Just The Peak Of Cell Growth, Because There Will Be Signal Pathways Between Cells And Each Other. At This Time, They All Agreed To Work Hard To Grow. After Passing On Generations, When They See That The Space Is Still So Large, They Will Pass On The Spirit Of Hard Growth Until They Pass The Peak Of Growth To The Peak Of Growth. Of Course, There Are Some Greedy Cells That Grow In Clusters. They Will Spare No Effort To Grow Day By Day Until The Medium Can Not Provide Them With Any Nutrients& Nbsp; Step 3: Important Passage Process That Cannot Be Ignored: 1.37 ℃ Water Bath Is Used To Heat The Complete Medium, 0.25% Trypsin/EDTA Solution. We Do Not Recommend Heating Reagents And Medium At 37 ℃, And Water Bath Is Preferred. 2.  Rinse The Cells With DPBS And Discard The Washed DPBS. 3.  Join 1; Ml 0.25% T/E Solution Into T-25 Bottle. Shake The Flask Gently To Ensure That The T/E Solution Completely Covers The Cells. The Morphological Changes Of Cells Were Observed Under Microscope. 4.。 During Incubation, Prepare A 15ml Conical Centrifuge Tube And 5ml Trypsin Suspension 5. During Digestion, Gently Tap The Side Of The Culture Bottle To Peel The Cells From The Surface. Check Under The Microscope To Ensure That All Cell Gaps Become Larger And Begin To Fall Off In Quicksand. 6..  Add 5 Ml Of Trypsin Suspension Into The Culture Bottle, And Transfer The Separated Cells Into A 15 Ml Centrifuge Tube. Clean The Culture Bottle With Another 5 Ml Of Trypsin Suspension To Collect The Remaining Cells. 7. Centrifuge 15 Ml Centrifuge Tube At 1000 Rpm For 5 Minutes To Collect The Digested Cell Precipitation. 8. Drop The Cell Sediment Into The Containers We Want To Separate Bottles To Complete Passage.