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Using Nb2a cells we will be studying at the cells how they survive in the different condition, how cells are differentiated, how cells are grown with the nutrients, and observing the cell morphology in the different condition. During this experiment there will be using different serum types will be used on the cells to grow. There will be different plates will be used to grow the cells like Poly-l lysine. Trypan blue dye will be used for the cell count. During this experiment cells will be trypsinized. By trypsinizing the cell, cell will be detached from the surface of the plate. Trypan blue enters the cell cytoplasm and makes the cell blue so that way it will be observed as a dead cell. Live cell has no outer membranes broken to dye to enter the cell. Live cells will be white colored. There will be two different condition will be performed. One condition is dbcAMP. It is a nonhydrolyzable analog of cyclic AMP it is also known to induce terminal differentiation in Nb2a cells. Second condition is performed on the Poly-l lysine. Poly-l lysine is used to coat the plates and cells will be grown on that plate and observed after 24hr and 48 hr.
Material and methods:
This lab was done in two different parts. Safety is first during this lab period. All the procedure was done in the hood. First step during this lab is that sprays down the hood with the 70% ethanol and turn on the hood fan. Spray down everything with 70% ethanol that is entering the hood. First students were given two plates of 35mm2 with NB2a cells by the instructor. First thing when students obtain the plates number the plate's plate #1 and 2 and observe the plate under the microscope and take a look at the morphology and confluency of the cell and recorded in to the notebook. Plates were taken in the hood to trypsinize the cell with the pre warmed trypsin. Before adding the trypsin to the plate, remove the medium from the plate very carefully with the fresh micropipette and discard it in the waste container. Using the fresh P1000 micropipettes carefully add the 1 ml of the serum free medium to wash the cell. Make sure that medium cover the all the part of the plate. To do that close the lead and swirl the plate so it covers all the parts of the plate and removes all the traces of serum from the plate that was in before. Take out the serum free media with the new pipette tip and discarded it in the waste container. Next step is to obtain the trypsin that is pre warmed. Using the new fresh pipette add the 1 ml of the pre warmed trypsin to both plates and incubate both plates for 5 to 7 min at the 37°C. By trypsinizing the cells it detaches the cell from the bottom of the plate and cells are swimming in the medium. While the plates are in the incubator take two micro centrifuge tubes and label it as before the plates were labeled. After incubation is done observe the plates under the microscope to see if the all the cells are detached from the bottom of the plates.
After the plates are incubated for 5 to 7 minutes take out the plates and observe the plates under the microscope to look at the cells if they are detached from the surface of the plates. If some cells are not detached than take the plates in to the hood and with the fresh pipette draw the medium and squirt it around the plates so cells are detached from the surface. Next step is to add the 1ml of Nb2a medium to each plate and slowly pipette the medium up and down to wash off all the cells in to medium. Transfer 1.5 ml from each plate to pre labeled centrifuge tubes. Close the cap tightly and centrifuge for 5 minutes at 4000rpm. After centrifuge is done take out the tubes and look for the white pallet at the bottom of the tube. Take out all the supernatant from the tubes and make and discard it in the waste container sure that you don't take the pellet out with the supernatant. Resuspend the pellet in 1ml of fresh growth medium. Now both tubes have concentrated cells in the tubes. Pipette the cells up and down so all the cells are mixed in the media. Transfer the 50µl from the each tube to new centrifuge tube and add the 10µl of trypan blue and 100µl of PBS. Close the cap and mix it well by shaking the tube. Now take 10µl from the tube and put it on the hemocytometer to count the live cells. Hemocytometer has two chambers you have to put the cells in to both chamber and then observe the cells under the microscope and look for the live cells (white cells). Blue cells are the dead cells. Collect cell count in to your notebook and calculate the cell concentration. With the remaining cell suspension create new plates for the following day experiment. Total there were 4 plates were plated with the cells 2 for 1% serum and 2 for Poly l lysine. Plated cells will be held in the incubation for the 24 hr and 48 hr.
There were two different condition was performed. Condition 1 is proliferation vs. dbcAMP- induced differentiation and condition 2 is Poly-L lysine and 1% serum. These both condition were two separate groups. My group performed Poly-l lysine and 1% serum condition. Second day 24 hour plates will be observed under the microscope and confluency will be noted in the notebook. Repeat the step for the trypsinizing the cells with the both condition plates. After trypsinizing the plates incubate the plates for 5 to 7 minutes. Take the plates out from the incubation and look at the cell under the microscope to make sure all the cells are detached from the plate surface. If cells are not detached from the surface with the fresh pipette draw the serum up and down several times. Next step is to count the cells so repeat the steps that were performed previous day for the cell count. Collect the data in to notebook and calculate the cells per ml. same steps will be performed for the 48 hr and cell count will be done. Calculate the cells per ml.
Table 1: Average for all the condition for 120 hrs
Poly- l Lysin
Graph 1: The graph above shows that how the cell amounts was changed in each condition at the different hours. It shows that in all the condition cells were growing until the 96 hours time period and then cells stated dying. This graph is average for all the lab sections. This graph compares all the condition with each other and shows how the cells survived in the each condition.
1st day cell count
Top = 61 x 1x104 = 6.1 x 105
Bottom= 63 x 1x104 = 6.3 x 105
Average = 6.2 x 105 cells/ml
After 24 hrs
1% Serum Poly- l lysine
Top = 4 x 1x104 Top = 15 x 104
Bottom= 5 x 10 4 Bottom = 6 x 104
Average= 4.5 x 105 cells/ml Average = 1.05 x 105 cells/ml
After 48 hrs
1% Serum Poly-l lysine
Top = 47 x 104 Top = 36 x 104
Bottom = 7 x 104 Bottom = 50 x 104
Average = 7 x 105 cells/ml Average = 4.3 x 105 cells/ml
As you can see in graph 1 that cells were reacted differently with all the conditions that we have preformed. 10% serum plates had the most cells count and the 1% serum had the lowest cell counts. There was change in the morphology of cells for all the different conditions. Condition that my group performed was poly-l lysine and the 1% serum. As you can see in the calculation that at the 24 hour cells did not grow that much at the 48 hour cells were more than before. Cells were in the incubation for enough time to get the all the nutrients and the factor they need to grow them. In table one 1% Serum cells number is getting lower as the hours are more and the dbcAMP condition the cells numbers are getting higher and at the 120 hrs cells starts to die.
During this lab we have learned many different ways to handle the cells, grow the cells, and how to transfer the cells in to another plates. The result for this lab was different for each lab section and each group, but there were some accurate result that we have expected for the lab. In this lab first part of the lab was to look at the cells and notice the confluency of the cells that are plated on the plates. We used the trypsin to adherent the cells from the surface of the plate and from each other. We use trypsin because it breaks down the protein in the cell and helps cells to separate from the surface of the plate. After all the trypsinization and incubation was done we counted the cells. First time when we counted the cells the cell count number was low so we counted second time and the number was little higher than the first time. The cell count number might be low because we didn't suspend the cells well and it didn't get mix well that might be the reason for the low number of cell count.
There were couple of different condition were done in this lab. My group did the 1% Serum and Poly- l lysine condition. To perform this condition cells were incubated for 24 and 48 hrs. At the 24 hour period we observed the both condition plates to look at the morphology of the cells and to look at if the cells have grown or died. First 24 hour plate had very little amount of cells were grown but at the 48 hour period cells were grown more than the before. Cells have grown more at the 48 hour because they were incubated more and they have been collecting all the nutrients and the growth factor they need to grow and the right temperature. Poly-l lysine promotes the cells to be adherence to plate and it might affect the proliferation.
Result for all the conditions was different from each other because all the condition works different with eh different type of medium and they might have different growing condition with the different nutrients and growth factor.