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

Question

Lab report is on Cell Staining

HELP PLEASE WITH CELL BIO LAB

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

1- Include a statement about the future scientific directions suggested by your findings from the experiment

2- After you have considered the data, write 1-3 sentences below that to summarize your interpretation of the findings.

Explanation / Answer

Wheat germ agglutinins (lectins) are versatile cationic probes for detecting glycoconjugates, i.e., N-acetylglucosamine and N-acetylneuraminic acid (sialic acid) residues, on cell membranes. Wheat germ agglutinin (WGA) bound prominently to glomeruli in all kidneys. Some of the experimental results shows, staining of the glomerular basement membrane with wheat germ agglutinin after trypsinisation significantly increases in the diabetic group when compared to both healthy and insulin-treated groups.

MitoTracker Green reacts with the free thiol groups of cystein residues belonging to mitochondrial proteins. Non-fluorescent in aqueous solution, becomes fluorescent once accumulated in the lipid environment of mitochondria. Permeabilization or fixation procedures cannot be applied to cells stained with MitoTracker Green. Using MitoTracker Green FM for mitochondrial mass estimation and Propidium Iodide for DNA content measurement.The co-localization of MitoTraker Green and TMRM in polarized mitochondria allows a fluorescence resonance energy transfer between these two dyes that give rise to red fluorescence emission with blue light excitation. MitoTraker Green is retained in the depolarized mitochondria, and its fluorescence emission becomes unquenched. MitoTracker Green, a probe used to evaluate mitochondrial mass, is a P-gp substrate, and its staining profile is dependent on the activity of this protein.

DAPI is generally used to stain fixed cells since the dye is cell impermeant. DAPI (4,6-diamidino-2-phenylindole) is a blue-fluorescent DNA stain that exhibits ~20-fold enhancement of fluorescence upon binding to AT regions of dsDNA. When DAPI binds with double strand DNA, the largest excitation wavelength is 360nm, while the largest emission wavelength becomes 460nm.

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