19.6.13
Date: 19.6.13
Time: 14:22
Location: Sir William Dunn School of Pathology, Oxford, England
This morning began with a trip to the cell bank. Stored in nine giant vats of liquid nitrogen, boxes and boxes of cells are kept at -198*C. The contents of each vat, which can hold around 90,000 cell capsules, are logged in a computer. (That's a maximum capacity of 810,000 cell capsules!) While this makes locating and organizing cells much easier, glitches in the system have electronically removed cells that are actually physically present. This then gives the cell bank moderator the difficult task of deciding whether to keep or dispose of unknown cells that can no longer be identified. Also, when placing the cells in the bank, an entire tray, which holds 8 8x8 boxes, must be removed from the jumbo in order to locate free space, raising the temperature of the cells and compromising the effectiveness of the liquid nitrogen cooling. (I see room for innovation!! Someone call the engineers!!)
Coming back from the cell bank I was put right to work preparing media in containers for cells to be defrosted after lunch. When cells get older, there is a much higher chance for mutation. Because of this, Helen B is tasked with keeping fresh, unchanged cells for the starting point for other people's experiments. Every once in a while she takes a few vials of cells and freezes them in the lab freezer at -80*C. She then takes them out later when needed and defrosts them and they "come back to life" and continue to split and live. However, the freezing and defrosting process is delicate because, if done incorrectly, can result in cell death, which could destroy the lab's supply of pure cells for experiments. If frozen too slowly or defrosted with too low a cell density or too much glycerol, the cells could die. Therefore, extra care is taken when dealing with freezing and unfreezing cells.
Watching another presentation to be given later in the week to the department by a member of the lab, it became clear that there are lots of little details and nit-picky things that must be done for a talk to be deemed ready, and even then there is always more work that can be done to refine the power point, talking points, or charts.
After lunch I ventured over to the Tissue Culture room to prepare slides to examine the bloodstream form of
the trypanosoma. First the medium was prepared, then the cells were diluted from an older sample to the newer samples. The newer samples were then placed in the incubator and the older ones used to prepare the slides. This way, Helen will have more cells for later experimentation because the cells will split in the incubator.
The other Helen was now prepared to defrost the cells in the freezer into the medium from the morning since they had now warmed to 28*C. The vials were taken out of the freezer and placed in a water bath at 28*C. After warming for just under a minute they were removed and flicked (yes, very scientific procedure, I know). They were then immediately pipetted in 1mL increments into the medium and then 1mL of this solution was placed into the other container of medium. This created one solution of concentrated cell 427 and one of 1/10th concentration. This provided enough space for the cells to move away from the glycerol, which they do not fancy very much once they are up and wriggling about again. From here the slides are prepared in the same way as before, rinsing in PBS and adding the appropriate antibodies and immunofluorescent indicators.
Finally, I learned about what another member of the lab is working on. By separating the strands of the genome, sections of RNA-i can be selected and chosen based on certain genes in the strand. The mRNA can then go in and destroy sections of the strand that are desired for the manipulation of the DNA of the cell. Once the RNA has been used to manipulate the genomes, the parasite cells are electrocuted, creating holes that the manipulated circular DNA from bacteria can then enter the cell and join the DNA of the parasite.
However this process only takes place in 1 in every 1000 cells but who wants to look at 1000 cells? Not I. So, in order to dispose of the cells that do not contain the altered genomes, a drug is added to the sample. This drug is tagged in the RNA-i and the cells that contain this tag are not affected by the addition of the drug. All the cells that do not contain the altered genes are killed by the drug, leaving a good sample of altered genes.
Time: 14:22
Location: Sir William Dunn School of Pathology, Oxford, England
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| Removing trays of boxes |
Coming back from the cell bank I was put right to work preparing media in containers for cells to be defrosted after lunch. When cells get older, there is a much higher chance for mutation. Because of this, Helen B is tasked with keeping fresh, unchanged cells for the starting point for other people's experiments. Every once in a while she takes a few vials of cells and freezes them in the lab freezer at -80*C. She then takes them out later when needed and defrosts them and they "come back to life" and continue to split and live. However, the freezing and defrosting process is delicate because, if done incorrectly, can result in cell death, which could destroy the lab's supply of pure cells for experiments. If frozen too slowly or defrosted with too low a cell density or too much glycerol, the cells could die. Therefore, extra care is taken when dealing with freezing and unfreezing cells.
Watching another presentation to be given later in the week to the department by a member of the lab, it became clear that there are lots of little details and nit-picky things that must be done for a talk to be deemed ready, and even then there is always more work that can be done to refine the power point, talking points, or charts.
After lunch I ventured over to the Tissue Culture room to prepare slides to examine the bloodstream form of
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| Bloodstream form of Trypanosoma Brucei |
The other Helen was now prepared to defrost the cells in the freezer into the medium from the morning since they had now warmed to 28*C. The vials were taken out of the freezer and placed in a water bath at 28*C. After warming for just under a minute they were removed and flicked (yes, very scientific procedure, I know). They were then immediately pipetted in 1mL increments into the medium and then 1mL of this solution was placed into the other container of medium. This created one solution of concentrated cell 427 and one of 1/10th concentration. This provided enough space for the cells to move away from the glycerol, which they do not fancy very much once they are up and wriggling about again. From here the slides are prepared in the same way as before, rinsing in PBS and adding the appropriate antibodies and immunofluorescent indicators.
Finally, I learned about what another member of the lab is working on. By separating the strands of the genome, sections of RNA-i can be selected and chosen based on certain genes in the strand. The mRNA can then go in and destroy sections of the strand that are desired for the manipulation of the DNA of the cell. Once the RNA has been used to manipulate the genomes, the parasite cells are electrocuted, creating holes that the manipulated circular DNA from bacteria can then enter the cell and join the DNA of the parasite.
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| Diagram of the RNA-i, mRNA and DNA process |
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