HELP PLEASE WITH CELL BIO LAB Lab report is on Cell Staining EXPERIMENTAL PROTOC

Question

HELP PLEASE WITH CELL BIO LAB Lab report is on Cell Staining

EXPERIMENTAL PROTOCOL

Using dyes that will differentially stain different organelles you could address basic questions regarding the composition (type and number) of organelles in cells, their enzymatic activity, health status, etc.

In this exercise, students will use Wheat Germ Agglutinin, MitoTracker Green and DAPI to stain the plasma membrane, mitochondria and nuclei of cells, respectively. Students will assess the appearance of these cellular stains in two different cell types and discuss their interpretations of the results obtained.

1/2 CLASS STAINED MONKEY KIDNEY CELLS AND OTHER 1/2 RAT CELLS

USED A FLUORENCE MICROSCOPE

1.) What cellular compartments are you staining in today’s lab session? Are you using chromogenic, fluorescence or antibody staining methods with your cells?

2.) Based on the chemical structure of each staining molecule, how and why do these dyes label a specific cellular compartment?

3.) Name the differences in terms of abundance (number) of MitoTracker Green FM positive structures among the two different types of cells. Did you notice any other staining differences between the cell types you examined? What physiological explanation might account for those differences?

4) Create a summary of the overall approach you use to answer the question being addressed.

Explanation / Answer

Question 1.The cellular compartments stained are a) Wheat germ agglutinin will stain the plasma membrane. Plasma membrane that separates the interior of the cell form the extra-cellular environment. It is mainly composed of lipid and protein molecules and form a barrier around the cell.

b) MitoTracker Green FM will stain the mitochondria. Mitochondria is a membrane bound organelle present in the cytoplasm of the cell.

c) The third compartment stained will be the nucleus using the DAPI (4',6-diamidino-2-phenylindole) stain.

All of the above dyes are fluorescent stains. The fluorescence staining is based on the principle of, a light of specific wavelength is absorbed by the specific fluorophore (stain) and light of longer wavelength is emitted.

Question 2.

a) Fluorescent lectins are versatile probes for detecting glycoconjugates and for localizing glycoproteins in gels. Wheat germ agglutinin has a molecular weight of 38000 daltons and exists as a heterodimer and is cationic under physiological conditions. These sialic acid rich glycoprotein residues are found on the plasma membranes of cells and they help keep water at the surface of the cells.

Wheat germ agglutinin is a stain that specifically binds to N-acetylglucosamine and N-acetylneuraminic acid (sialic acid) residues. The carbohydrate-binding specificity of wheat germ agglutinin is directed against sequences of -1,4- N-acetylglucosamine linked residues – the chitodextrins.

b) Mito Tracker Green FM in nanomolar concentrations stains the mitochondria in the cells. It exhibit bright green, fluorescein-like fluorescence and labels mitochondria within live cells utilizing the mitochondrial membrane potential. It is chemically reactive, linking to thiol groups in the mitochondria. The dye becomes permanently bound to the mitochondria. The MitoTracker Green FM dye has the added advantage that it is essentially nonfluorescent in aqueous solutions and only becomes fluorescent once it accumulates in the lipid environment of mitochondria.

c) DAPI is a blue-fluorescent DNA stain and binds strongly to A-T rich regions in DNA. The mechanism of DAPI-DNA binding is not by intercalation. The binding geometries and site densities are consistent with DAPI located in the grooves of DNA, with the high-affinity site probably in the minor groove of DNA that contain strong A-T rich regions.

Question 3. Monkey kidney cells contain > 2000 mitochondria per cell compared to the rat cells. Hence, the monkey kidney cells show higher green fluorescence as compared to rat cells using the stain. No other differences in staining were observed. As kidneys need very high source of energy and is only second in number to heart in mitochondrial number. As mitochondria is considered a power house, generating a large source of ATP their number is high in the kidney cells. The proximal tubes of kidneys use the majority of oxygen for ATP generation and hence contain most number of mitochondria.

Question 4. The summary for the approach is as follows. First the understanding about the cell organelles is being presented, followed by explanation of the detection method used In the next question, the specific mechanism for the staining is being explained. In the last question, the difference between the two cell types and the reason why kidney cells contain more mitochondria is being presented.

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