Cell Culture Errors

• Trypsin is used in cell passaging and cultures to detach cells from the surface of the cell culture media.
• If cells incubate with trypsin for too long, they become enlarged and more circular, excessively degrade, surface proteins get damaged, and may lead to cell death.
• A scientist fixes breast cancer cells with 30% paraformaldehyde and incubates for 12 hours.
• Paraformaldehyde freezes the cellular structure but long incubation alters the native protein structure of the cells.
• Breast cancer cells are placed in an incubator and the temperature drops by 7 degrees overnight.
• Changing the optimal conditions alters cell homeostasis and the cells spend energy trying to adapt, resulting in less cell division.
• The cells may die if the temperature is too low.
• A student obtained 1000 ng/microliters of RNA with a 260/280 ratio of 1.1, which is low.
• The low ratio implies protein contaminants are in the solution, since pure RNA would have a ratio greater than 1.8.
• To label two proteins (actin and tubulin) within the same cell, the immunofluorescent technique is used.

Immunofluorescent Technique

• First, a primary antibody specific to actin is used.
• A secondary fluorescent antibody binds to the primary antibody to visualize actin.
• A different primary antibody specific to tubulin is used with a secondary fluorescent antibody to visualize tubulin.
• Different color antibodies are used to emit different wavelengths that are distant from each other and do not overlap, so there is no fluorescence.
• Filters are used for each color to visualize and distinguish between actin and tubulin under a fluorescent microscope.
• Cytochalasin B interferes with actin dynamics by blocking the positive end and preventing polymerization.
• The morphology becomes rounded and irregular because actin filaments depolymerize and lose structural integrity.
• In a real-time PCR amplification curve, the plateau phase means there is no further DNA amplification because all the resources have been depleted.
• In a real-time PCR experiment, if you obtain 1 million copies in cycle 25:
• In cycle 24, there would be half, which is 500,000 copies.
• In cycle 26, there would be double, which is 2 million copies.
• In a real time PCR experiment, SYBR Green is a fluorescent antibody that binds to doubled DNA, making it visible and fluorescent.
• SYBR green gives an indication of how much DNA has been amplified since it only fluoresces when bonded.

SYBR Green

• By counting the number of SYBR green antibodies, the DNA copy number is quantified.
• Increasing the gain on the fluorescent image amplifies the signal, making it more visible.
• Photomultiplier tubes are highly sensitive light detectors used in fluorescence microscopy.

Process of Blocking

• All potential antibody binding sites in the sample are blocked by serum.
• This will allow the antibody to bind to its target protein and not other proteins, increasing the background signal.
• The blocking solution covers the nonspecific proteins so that the specific antibody can only go to the specific protein.
• Fluorescence microscopes are great for quick and simple imaging but struggle with out-of-focus light.
• Confocal microscopes provide sharper, high-resolution images by eliminating background fluorescence, making them ideal for thick samples and 3D imaging.
• Fluorescence is more affordable while confocal is more expensive.
• Permeabilization is used to target a protein in the cell by adding antibodies to make the membrane more permeable.
• Reagents such as Triton X disrupt the lipid membrane by creating pores that allow the antibodies to penetrate the cell and bind to intracellular proteins.

Targeting Mitochondria Using Fusion Proteins

• First, design a fusion protein by attaching a mitochondrial targeting sequence (MTS) to a fluorescent protein (e.g., GFP, RFP).
• Clone the construct into an expression vector and transfect or transduce live cells.
• Allow 24-48 hours for protein expression.
• Optionally, stain with a mitochondrial dye (e.g., MitoTracker) for colocalization.
• Image the cells using fluorescence or confocal microscopy.
• Analyze mitochondrial localization and dynamics to confirm proper targeting.

Common Obstacles in Fluorescence Microscopy

• Bleed through: when GFP emission overlaps with YFP emission, giving false results.
• Background noise and non-specific signals: non-specific bindings to antibodies.
• Bleaching and weakening of signals: Fluorophores are highly light sensitive and exposure of those fluorescent molecules to light cause bleaching of the fluorophore. Weak signals could disappear before detection, resulting in misinterpretation of the results.
• Focusing of image.