Practical Operation RAW264.7 Cell Passage Treatment

1、 Background: RAW264.7 Cells Are Derived From Tumor Tissue Induced By Abelson Mouse Leukemia Virus, Which Is One Of The Most Commonly Used Inflammatory Cell Models In Scientific Research. This Cell Is Easy To Proliferate, Has High Efficiency Of DNA Transfection, And Is Sensitive To RNA Interference. It Is Often Used To Transfect Host Cells And Replicate Mouse Norovirus. The Cells Were Negative For SIg -, Ia - And Thy-1.2 Antigens. It Was Reported That The Cells Did Not Secrete And Virus Particles Could Not Be Detected After The Establishment Of The Cell Line, And The XC Spot Formation Test Was Negative. It Can Decompose Sheep Red Blood Cells And Tumor Target Cells In An Antibody Dependent Manner. LPS Or PPD Treatment Can Induce The Decomposition Of Red Blood Cells After 2 Days, But Has No Effect On Tumor Target Cells. 2、 Morphological Characteristics Of RAW264.7 Cells 1. RAW264.7 Cells Are Generally Round Or Oval, With Round Or Oval Nuclei And Deep Staining. The Wall Is Especially Firm. If The Cells Are Polygonal, They Are Activated. 2. RAW264.7 Is Capable Of Phagocytosis. When Cells Phagocytosis Antigens, The Cells Will Release Chemokines, Further Promote The Cells To Extend Pseudopodia, And Enhance The Ability Of Adhesion And Climbing. If The Cells Swallow Too Many Antigens, The Culture Environment Is Bad, And The Cells Are Relatively Sparse After Passage, The Cells Will Be Spindle Shaped And Long Spindle Shaped. Under The Microscope, Small Cells With Slender Pseudopods Will Be Covered By Spindle Cells Similar To Epithelium. 3. This Cell Belongs To The Slow Heat Type And Is Difficult To Digest. If The Digestion Time Is Not Enough, The Cell Will Not Be Able To Digest. If The Digestion Time Is Too Long, After Passage, It Will Grow A Horn For You To See, Blow Hard And Scrape Off The Cell Knife. After Passage, It Will Not Only Grow A Horn For You To See, But Also Float A Lot Of Small Black Spots. Moreover, The Inoculation Density Of This Cell Should Be Large When Passage, Otherwise It Is Easy To Differentiate Macrophages And Deform Cells, And It Can't Recover, Which Is The Most Important Problem To Pay Attention To When Cultivating Such Cells. 4. RAW264.7 Cells Like Company When They Grow. The Normal Growth State Should Be The Growth Of Small Pieces. The Pieces Are Not Connected. When They Grow More And More Densely, They Will Be Connected Into Large Pieces And Stacked Into Layers. The Stacked Cells Need More Nutrition. Once The Nutrition Cannot Keep Up, The Cells Will Also Grow A Corner For You To See, So You Need To Operate When The Cells Are Stacked. 5. RAW264.7 Cells Adhere To The Wall Quickly After Passage, And Can Attach A Large Area Within Half An Hour. When They Just Adhere To The Wall, The Cells Are Round With Good Refractive Index. Under The Microscope, They Are Scattered On The Bottom Of The Bottle Like Small Pearls. If You Feel That There Are Many Activated Cells, That Is, There Are Many Long Spindle Cells, You Can Change The Fluid Half An Hour After The Cells Pass On, Digest And Adhere To The Wall Again, And Only Keep The Cells In Good Growth Condition. 6. The Growth Of RAW264.7 Cells Requires A High Level Of Serum, And Poor Serum Will Lead To Poor Cell Growth. This Is A Big Stomach King, So Don't Hesitate To Change The Liquid Immediately When You See The Medium Turning Yellow. 3、 Precautions In Culture And Passage Step 1. Before Culture, All Glassware And Utensils In Contact With Cells Should Be Heat Treated. (Baked At 200-250 ℃ For 4-6 Hours) Our Digestion And Passage Method: Method 1. Digestive Solution: 0.5% Trypsin, 0.2 EDTA, Heated To 37 ℃ Before The Experiment. Take T25 Culture Bottle As An Example: 1. When The Cells Adhere To The Wall And Grow To 95% Of The Bottom Of The Bottle, Discard The Culture Solution, And Add D-HANKS To Rinse Twice& Nbsp; 2. Add 1-2ml Of Digestive Juice Of Trypsin And EDTA, Digest At 37 ℃ For 2-5min, And Under Microscope, It Can Be Seen That Cells Basically Fall Off, That Is, Digestion Is Terminated, And The Digested Cells Are Collected; (Because The Cells At The Bottom Usually Have Pseudopods, Which Are Tightly Attached To The Bottle Wall, It Is More Difficult To Digest. When Digesting, You Can Collect The Cells At The Top, And Then Digest The Cells At The Bottom. The Cells Digested Each Time Cannot Be Put In The Same Bottle.). 3. Add D-HANKS For Rinsing Twice, Add 1-2ml Of Trypsin And EDTA Digestive Solution, Digest For 2-5min At 37 ℃, Continue To Digest The Cells At The Bottom Layer, And Stop Digestion When Cells Fall Off Under The Microscope. Collect The Digested Cells Separately; 4. Add 10Ml Of New Culture Medium And Gently Stretch The Suspension Cells. Divide The Suspension Cells Into New Culture Bottles As Required. In The 37 ℃ CO2 Incubator, The Suspension Cells Can Adhere To The Wall In A Few Hours. 4、 Precautions For Freezing: 1. The Freezing Process Of RAW264.7 Should Minimize The Impact Of Heat Source On Cells, So As To Better Ensure The Cleanliness Of Cell Seeds. 2. This Kind Of Cells Is Very Sensitive To The Space State, So The Density Of The Recovered Cells Or The Number Of The Recovered Cells Should Be Controlled At 10 ^ 4-10 ^ 5. The Density Of The Cells Can Reach 70-80% After This Inoculation Density Is Inoculated In A 75cm2 Culture Bottle For 2 Days. If The Cells Have Pseudopodia During The Recovery, Let The Cells Grow To A Stack First, And Then Pass On. Discard The Cells At The Bottom, And Try To Select A Good Serum For Culture, The New Cells Can Slowly Reach The Inactive State.