Visualization Dapi Rhodamine.docx

DEMONSTRATION ON VISUALIZATION OF SUB-CELLULAR ORGANELLES USING FLUORESCENT DYES

1. INTRODUCTION a) OBJECTIVES:

i) To visualize sub cellular organelles and nucleic acid using fluorescent dyes. b) PRINCIPLE: DAPI Staining: DAPI (diamidino-2-phenylindole) is a fluroscent stain that binds strongly to the DNA. It is rapidly taken up by the cellular DNA and selectively binds to the minor groove for doube stranded DNA. The excitation maximum for DAPI bound to dsDNA is 358 nm, and the emission maximum is 461nm. DAPI can be used for both fixed and live cell staining. Rhodamine 123 Staining: Mitochondria exist in most eukaryotic cells and play a very important role in oxidative metabolism by generating ATP as an energy soruce. The average number of mitochondria per cell is from 100 to 2,000. Since mitochondria have electron transport systems, they can be stained with various redox dyes. MitoRed and Rh123 readily pass through cell membranes and accumulate in mitochondria. The fluorescence intensity of Rh123 reflects the amount of ATP generated in mitochondria. Rh123 is a lipophilic cation. The transmembrane potential (inside negative) of the mitochondria may be responsible for the uptake of R123.

Fig 9.1. Cell Staining Methods

Target

Dye

Excitation

Emission Excitation filter

Colour

Characetristic

Dead Cells

DAPI

360nm

460nm

blue

Fluorescence is produced by

W excitation

Mitochondria Rh123

2. ACQUISITION

507nm

529nm

G excitation

red

interacting with the nucleus of dead cells Flourescenc is produced by accumulation in mitochondria

:

a) MATERIALS REQUIRED: 1. 0.01 M Phosphate Buffer (137 mM NaCl, 2.7 mM KCl, 10mM Na2HPO4, 1.8 mM KH2PO4, 1mM CaCl2, 0.5mM MgCl2, pH 7.4) 2. DAPI 3. Rhodamine 123 (brown powder avoid light, refrigerate) 4. Poly-L-Lysine pre-coated slides b) PROTOCOL (STEP WISE): 1. Cells were harvested at stationary phase (normalized OD of 1.0) by centrifuging at 14000 rpm for 2 min, the culture media was removed, and the pellets were washed once with 1mL of 0.01M Phosphate buffer (PB). 2. Fixation: i) Cell pellets for staining were then fixed in 1:3 ratio of PB and 4 % paraformaldehyde for 20 minutes at 4 deg C. ii) Slide Washing: Place the Poly-L-Lysine pre-coated slides on a clean tissue paper. iii) Gently flood the slide first with 70% ethanol (filtered) and then with DI water (filter serile). iv)Dry the slides in hybridization oven for 5 minutes. 3. Smearing: i) Draw a well for smear and maintain the inner diameter of approximately 1 cm. ii) 25ul of fixed cells was gently smeared on to Poly-L-Lysine pre-coated slides and allowed it to air dry at room temperature for 10 minutes and then at hybridization oven. 4. DAPI staining: DAPI staining was achieved by adding 1ul of DAPI stock (10 mg/ml) to 1 ml of the diluted cultures to give a final DAPI concentration of 10ug/ml. Stained bacterial cultures were collected by centrifuging (13,000 g for 2 min), washed twice with filter sterilized Phosphate Buffer Saline (PBS) and finally resuspended in 1 ml of filter sterilized PBS. 5. Mounting the coverslip and Imaging: For microscopic observation, 10ul of each mixture was mounted onto microscopic slide and observed under Olympus Inverted phase contrast for the presence of fluorescence.

b) Rhodamine123 staining: 1. Preparing the stock solutions for the fluorescent probes Rh123: Prepare a 10-mM stock solution of Rh123 in 1 ml of anhydrous DMSO. 2. Remove the supernatant of the media, and add PBS (-). At this step, adjust the cell volume to 5 X 108 cells/ml. 3. Add 200 µl of the cell suspension to a microtube. 4. In order to permeabilize the bacterial outer membrane, EDTA was added prior to the dyes to a final concentration of 1 mM 4. Add 100 µl of Staining solution to the same tube. 5. Incubate at 37 o C for 15-30 min with protection from light. 6. Place 10 µl of the cell and staining solution on a glass slide and cover with a cover glass and observe under microscope. d. RESULT

e. INFERENCE:

3. PRACTICE / TESTING: Questions and Answers: