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What is Formaldehyde used for and how does it work?
Formaldehyde is used for cellular fixation.
more info: http://tinyurl.com/mzrq4pz
- Formaldehyde causes fixation by cross-linking together primary (1o) amine groups of proteins and nearby Nitrogen atoms in other proteins or DNA. Formaldehyde links these two together through a -CH2- linkage (provided by formaldehyde). This fixation can help stabilize the structure of a cell ("freezing" it as it were at the time of fixation), or it can affect the charge at the site of attachment.
- >> since formaldehyde reacts extensively with amino groups [to form methylene bridges], it can/will essentially negate the positive charge that these amines would normally have conferred for chemical reactions, and thus these groups are no longer available to bind to negatively charged molecules, for example, the dye molecule eosin.
What is PBS (Phosphate Buffered Saline)?
PBS is a buffer that has osmolarity and ion concentrations that match those of the body (and is also isotonic like the body).
- Things that may typically be found in PBS include:
- Sodium Phosphate
- NaCl (Sodium Chloride)
- KCl (Potassium Chloride)
- Potassium Phosphate
- pH = 7.4
- *some formulations may also contain Calcium or Magnesium
How is PBS used?
Since PBS is isotonic and non-toxic to cells, it has many uses. Namely, these include substance dilution and cell container rinsing.
What key things are contained in the Growth Media that we use (DMEM)?
>> "Dulbecco's Modified Eagle's Medium"
DMEM is a modified/improved
version of the original "EMEM" (Eagle's minimal essential medium - developed by Dr. Eagle):
- - Amino Acids (4x the original)
- - Glucose (2-4x the original)
- - Vitamins (4x the original) (B Vitamins)
- - Salts
- - Iron (not found in original)- Phenol Red (not found in original) (is a pH indicator; gives the distinct color we know it by)
How does CAM (Calcein Acetoxymethylester) work?
CAM can readily penetrate healthy cells. Once entering a healthy cell it is rapidly hydrolyzed by intracellular esterases, losing it's acetomethoxy group to become polyanionic Calcein - which is intensely fluorescent (green) and well retained in living cells (additionally because it has such a negative charge from being polyanionic).
This is useless to dead cells since dead cells don't have active esterases that can hydrolyze the CAM to yield it's fluorescent breakdown product. Thus, CAM is only used to tag living cells.
How does EthD (Ethidium Homodimer) work?
Normally, Ethidium Homodimer is not membrane permeable.
However, when cells die, the plasma membranes of those cells becomes disrupted. Because of this, Ethidium Homodimer may then enter those cells and bind to DNA within those cells, whereafter it will fluoresce red. Because live cells don't have a compromised membrane, the Ethidium Homodimer can't enter.
How does Trypan Blue work?
Trypan Blue is used to selectively color dead cells.
Since live cells still have intact membranes, which are highly selective on what's allowed to pass through, Trypan Blue can only penetrate the membranes of dead cells (since upon death, they become disrupted, leaky, and penetrable). Upon traversing a dead cell's membrane, Trypan Blue simply stains the cell it's signature blue color, and thus shows up as a dark blue in cultures. Thus, any cell that does not appear dark blue (i.e. just ambient-colored blue) is in fact, still a live cell.
How does TrypLE work?
TrypLE is used for the dissociation of attachment-dependent cell lines from plasticware so that the cells can be suspended in fresh solution and transferred to fresh dishes. It works by cleaving the peptide bonds on the C-terminal sides of Lysine and Arginine residues (making it a direct replacement for trypsin). TrypLE is also less aggressive than regular trypsin, which makes it gentler on cells.
How are Monoclonal Antibodies produced?
- Monoclonal Antibody production:
- First, an antigen is injected into an organism's (e.g. Mouse) bloodstream. The mouse's immune system B-Cells then respond by eventually recombining to one that produces an antibody that can bind to the injected antigen. With a B-Cell present that has figured out a way to attack this foreign antigen, the body of the Mouse then makes MANY copies of this specific B-Cell, giving rise to a line of clones, each of which is genetically capable of producing the antibody for the injected antigen. Serum is then drawn from the Mouse and presented into a solution containing our desired antigen (the one we wish to bind to or take out). If a B-Cell is present in the serum that can bind to the antigen then it will precipitate with it (if nothing binds then we wait more time until we draw serum again, hoping the mouse by then has produced B-Cells for the antigen, repeat). The spleen of the mouse is then taken out and dissociated into a culture medium to release the resident B-Cells; the culture medium also contains cells from a special myeloma cell line in it (special = can reproduce indefinitely but do not produce antibodies). Then, PEG (Polyethylene Glycol) is added to the mixture, and some of the Myeloma cells go on to fuse with the B-Cells, forming hybridomas. Then, the cells are placed in HAT medium (Hypoxanthine-Aminopterin-Thymidine medium) - in which any unfused cells eventually die, leaving only hybridomas left. Now, while only hybridomas with the genetic capacity to produce antibodies for our antigen should be left, some others may have slipped through and so we need to screen them. Screening is done by diluting and literally plating just 1 cell per well on a microplate. The cells are given a few weeks to grow, and are then presented with our specific antigen that we wish to target. THEN, only the cells from wells that responded to our antigen will have their cell lines expanded in vitro by placing them in a culture flask containing fetal bovine serum (trying to produce them in mass in vivo however is known to produce other random artifacts/impurities, and so in vitro is the preferred method).
- Tada! A new Monoclonal Antibody is produced!
What is Confluency?
Confluency = the percentage of growth surface covered by cells
What does Hematoxylin do?
What color does it produce?
- Hematoxylin stains NEGATIVELY charged molecules blue
- >> so, Hematoxylin itself is a positively charged dye
What does Eosin do?
What color does it produce?
- Eosin stains POSITIVELY charged molecules red
- >> so, Eosin itself is a negatively charged dye
When tagging (aka "conjugating") an Antibody, what is important to factor in when picking your tag?
- That what you are tagging it with is detectable
(i.e. fluoresces, luminesces, absorbs light [like a heavy metal] if doing electron microscopy, etc.)
- - That what you are tagging it with would not normally be there [in that cell]
- >> ex: Horseradish Peroxidase
What is a key advantage of using fluorescence?
Why is it better than regular luminescence
- Fluorescent molecules give off fresh, very high-energy light! This light is much more intense (has more energy) than regular light that we would receive reflected off of an object (ex: would be brighter than the blue light reflecting off of a blue shirt) since this fluorescent light is newly created!
- >> This new light, being much brighter, gives us much better contrast!
What is a downside of using immortalized cell lines? Why is this the case?
Eventually, after enough time, immortalized cells start to lose their cellular responsiveness to hormones, other cells, etc.
This is the case because by living in vitro for so long, these cells simply haven't needed to respond to these things since they never received a stimulus (ex: by a hormone) to do so! So they've forgotten how to respond now, even if re-presented with the stimulus again. Thus, "this" response pathway has now atrophied.
What is a big advantage of working on something in vitro instead of in vivo?
- By studying something in vitro you no longer need to worry about maintaining and considering the possible alternative explanations provided by things like:
- - pH, Temperature, Hormones, Immune System, etc.
Because of this, you can be significantly more certain that if you observe a response in a specimen, that it was caused by what you did
and not by the environment
What do Loss-of-Function experiments allow you to do? (i.e. why would you do these)
Loss-of-Function experiments allow us to see what defects are caused by the mutation/knockout of the gene and, thereby determine the role of the particular gene