Propidium Iodide Solution

Transcription

1 G-Biosciences A Geno Technology, Inc. (USA) brand name Propidium Iodide Solution (1mg/ml in deionized water) (Cat. # , ) think proteins! think G-Biosciences

2 INTRODUCTION... 3 ITEM(S) SUPPLIED... 3 STORAGE CONDITIONS... 3 WARNING... 3 SPECIFICATIONS... 3 ADDITIONAL ITEMS REQUIRED... 4 PROTOCOL... 4 REFERENCES... 6 RELATED PRODUCTS... 6 Page 2 of 8

3 INTRODUCTION Propidium iodide (PI) is a red fluorescent cell viability dye which is excluded from live cells as it is impermeable to the cell membrane. The dye intercalates DNA and RNA of the dead cells to give red fluorescent signal as it can penetrate dead or damaged cells to give red fluorescent signal. The fluorescence of the PI dye is enhanced 20 to 30 fold upon binding to the nucleic acids and the fluorescence excitation maximum is shifted ~30 40 nm to red and the fluorescence emission is shifted ~15 nm to the blue 1. Propidium iodide is used widely as a counterstain to differentiate and exclude nonviable cells in flowcytometry analysis. It is used in DNA fluorescence imaging to discriminate early and late stages of apoptosis, used to study cell mediated cytotoxity and for chromosome analysis. It is also used in quantitative DNA analysis. ITEM(S) SUPPLIED Cat. # Description Size Propidium Iodide Solution 2 ml Propidium Iodide Solution 10 ml STORAGE CONDITIONS Propidium Iodide Solution is supplied at ambient temperature. Upon arrival store at 4 C. The product is stable for 1 year when stored as instructed. WARNING Propidium Iodide is a potential mutagen. Handle the dye with care and dispose off the waste safely as per applicable local regulations. SPECIFICATIONS Fluorescent dye, membrane impermeable nucleic acid intercalator λex/λem (DNA bound) = 535/617 m λex/ λem (no DNA) = 493/636 nm Form: solution, sterile filtered Concentration: 1mg/ml in deionized water Storage: 4 C; protect from light Molecular formula: C 27 H 34 I 2 N 4 Molecular weight: Cas number: Page 3 of 8

4 ADDITIONAL ITEMS REQUIRED Fluorescence Microscopy 20X SSC (0.3M NaCl, 0.03M sodium citrate, ph7.0) (Cat. # ) LongLife RNase (Cat. # ) QuenchShield Mountant (Cat. # ) Flow Cytometry PBS Ethanol Staining Buffer (100 mm Tris, ph:7.4, 150mM NaCl, 1 mm CaCl 2, 0.5 mm MgCl 2, 0.1% Nonidet P 40) Chromosome FISH Deionized water PBS LongLife RNase (Cat. # ) QuenchShield Mountant (Cat. # ) PROTOCOL Fluorescence Microscopy Protocol for counterstaining cells with propidium iodide 1. Fix the cells as per your sample. 2. Perform all other staining as per your standardized method. NOTE: PI staining is performed after all other staining. 3. RNase treatment: a. Dilute 20X SCC to 2X by mixing 20X SSC with deionized water in ratio 1:19 b. Equilibrate the samples with 2X SCC. NOTE: RNase treatment is required for samples fixed with paraformaldehyde, formaldehyde or glutarldehyde. NOTE: RNAse treatment is not required for samples fixed with methanol/acetic acid or acetone. c. Incubate the sample in 100 µg/ml LongLife RNase in 2X SCC for 20 minutes at 37 C. Page 4 of 8

5 d. Rinse the samples three times in 2X SSC for 1 minute each. 4. PI staining : a. Equilibrate the sample with 2X SSC. b. Prepare a 500 nm working stock of propidium iodide in 2X SCC by adding propidium iodide solution to 2X SCC in ratio 1:3000. c. Add µl of working stock of PI on sample to allow cell or tissue to completely submerge in PI. Incubate the slide/coverslip for 5 minutes. d. Rinse the sample on slide/coveslip with 2XSCC several times. e. Drain excess buffer with kimwipe and add drop of QuenchShield Mountant on slide/coverslip to mount the sample. NOTE: For sample on coverslip add a drop of mountant on coverslip and flip the coverslip on a glass slide. For sample on slide add a drop of mountant on sample on the slide and gently place a coverslip avoiding any air bubbles trapped between coverlip and slide. f. View sample under fluorescence microscope adjusted at appropriate filters. Flow cytometry Protocol for counterstaining suspended cells with propidium iodide 1. Collect volume of suspension equivalent to 2 x 10 5 cells to 1 x 10 6 cells. 2. Pellet the cells at 500 g for 5 min. 3. Discard the supernatant and tap the tube to loosen the cells in residual liquid. 4. Add 1 ml PBS and resuspend cells. 5. Transfer the cells slowly by using a pipette to 4 ml chilled ethanol (tube kept earlier in freezer) while vortexing. 6. Leave the cells in ethanol at 20 C for 15 minutes. 7. Pellet the cells at 500 g for 5 minutes, discard ethanol, tap the tube to loosen cells and then add 5 ml PBS at room temperature. 8. Resuspend the cells gently and incubate the tube at room temperature for 15 minutes to allow cells rehydration. 9. PI counterstaining: a. Prepare 3 µm working solution of PI in Staining Buffer by adding Propidium Iodide Solution to Staining Buffer in ratio 1:500. b. Centrifuge the cells suspended in PBS at 500 g for 5 minutes. c. Discard the supernatant, loosen the cells by tapping the tube and then add 1 ml of working solution of PI and resuspend cells. d. Incubate cells in PI for 15 minutes at room temperature and analyse the cells by flowcytometry in the presence of dye. NOTE: PI fluorescence is detected in the FL2 channel of Flow Cytometers. Chromosome FISH: Counterstaining specimen with PI. a. Prepare the specimen according to the standard procedures 2. Page 5 of 8

6 b. Before counterstaining, rinse the specimen with deionized water to remove excess salts. c. Prepare 1.5 µm PI staining solution by adding Propidium Iodide Solution to PBS in ratio 1:1000. d. Add 300 µl staining solution directly on the specimen. Add RNase to a final concentration of 10 µg/ml. e. Incubate the specimen in dark for 30 minutes at 37 C. f. Rinse the slide with PBS or water to remove dye. g. Use kimwipe to remove excess water. h. Add a 1 2 drop of QuenchShield Mountant to the sample. Gently cover the sample with coverslip avoiding air bubbles. i. Remove excess QuenchShield Mountant with kimwipe avoiding any air bubbles between slide and coverslip. j. Seal the edges of coversllip with wax or nailpolish. k. View sample under fluorescence microscope adjusted at appropriate filters. REFERENCES 1. Donna J. Arnt Jovin and Thomas M. Jovin (1989). Fluorescence Labeling and Microscopy of DNA. Methods Cell Biology Hames, B. D. and Higgins, S. J. (1985). Nucleic acid hybridization, A Practical Approach. RELATED PRODUCTS Download our Protein Labeling Conjugation Handbook. protein labeling conjugation handbook/ For other related products, visit our website at or contact us. Page 6 of 8

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