Monthly Archives: July 2012

7.19.12 Seeding cells on the scaffolds

Great news! The mouse osteoblasts are ready to be seeded on the scaffolds  Bryan Baker was kind enough to give me 400,000 hBMSC cells that he had cultured already. So I’m off and running on the second trial of this experiment! Here’s how I seeded the cells. Here’s the set up: Next I needed to label the plates I would be using to hold the scaffolds, one plate for 18 scaffolds for the mC3T3-E1 cells, one plate of 18 scaffolds for the hBMSC-8001 cells and one plate of 6 scaffolds, 3 for each cell type to be run for 14 days.     First I transferred the scaffolds to a sterile plate and then checked to make sure the curved side of the scaffold was facing down:   Next I need to add media to all the wells being careful to use the appropriate type of media for the different cell types:   . Next I used the vacuum to force the media into the cell holes: Then I was ready to remove 100 uL of media from each of the wells for the hBMSC-8001 cells and place it in the waste bucket: .Then I added 100 uL of the hBMSC-8001 cells to each of the 18 plus 3 wells with scaffolds . Once the cells were plated it went into the incubator. Now  needed to free the mC3T3-E1 cells that had adhered to the flask. First I transferred the media to the waste container:   Then I added a Trypsin solution to the flask that helped free the cells from the flask After 5 minutes I knocked the flask to free more cells then collected the solution: . I needed to centrifuge the solution to create a cell “pellet” so that I could count the number cells in the solution:  Notice the white pellet at the bottom of the tube? I then poured off the supernatant (solution floating above the pellet): I then added 2 mL of media and mixed up the cells to determine the concentration using flow cytometry (see 6-26-12-seeding-the-cells-on-the-scaffolds for a discussion of how this works: I found that there were 2,100,000 cells in my 2mL sample but I only needed  concentration of 600,000 in 3 mL. I used the formula C1V1=C2V2 where C1= concentration 1, v1 = volume 1, c2= concentration 2, and v2= volume 2. Here’s where I used my algebra skills to plug in my known values to determine how much volume I needed to add to my media solution to get the correct concentration. 2,100,000 x ? = 600,000 x 3 mL

2,100,000 x ? = 1,800,000

? = 1,800,000/2,100,000

? = 0.857 mL . That means I needed to transfer 857 uL of cells solution to 2.143 mL of media to make final solution of 3 mL with a concentration of 200,000 cells. Then I  needed to add 100 uL of this solution to each of the 21 scaffolds for the mC3T3-E1: . These plates also got incubated for 24 hours as well. Up next Day 1 fluorescence imaging and freezing picogreen samples.

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7.17.12 Changing Cell Culture Media

The cells that are used in the cell culture have been frozen in liquid nitrogen for long periods of time. In order to make sure the cells do not expand and burst when the freeze a detergent is added to the solution to poke holes in the membrane. This allows the cell to expand as the liquid inside changes into a solid. When the atoms become more tightly compressed and aligned, they increase in volume. These little holes allow the cell membrane to stretch instead of burst. That’s fantastic for when the cells are frozen but not necessary when the cells are at room temperature. To get rid of the detergent I needed to change the media 24 hours after I started the culture.Here’s how I set up my station under the hood: While I set up my station the media I used needed to heated to 37 degrees Celsius, the same temperature as the incubated cells. Once I replace the old media I went to check the cells under the microscope and this is what I saw: Notice how man of the cells have changed their shape (morphology) and spread across the surface of the flask? That means the cells were growing well and would be ready to seed on the scaffolds on Thursday.

Gel Electrophoresis Demo for NIST Summer Institute

Below are the images I took as I set up the gel electrophoresis lab. Remember to try it first before doing it for the students.  Play around with the staining times to see what gives you the best images. Let me know if you have any questions!

       hints-and-suggestions-for-gel-electrophoresis

7.16.12 Making Media and Growing Cell Culture for Trial 2

Hello! One of the things we discuss in class is how can we make sure that our results from an experiment are accurate? Do the experiment again!

So that’s what I started today. First I needed to make new media for the cells since this media includes proteins that breakdown over time. Here’s how I set up my station: The work needed to be done under a sterile hood since I do not want any contamination in the culture. I used an automated pipette to transfer 45 mL of media to two empty tubes:   The pipette can transfer 10mL at a  time. How many times should I have transferred the media and how much should I transfer at a time? Next I needed to add 5mL of fetal bovine serum to each vial of media:    Then I added 300 uL of antibiotic to the media as a preservative:   . Now I was ready to start the cell culture. I transferred 10 mL of media to the flask: then I waited for the frozen cells to thaw: then transferred the 1.7 million cells in 1 mL solution to the media in the flask: . And finally I checked the cells under the microscope at 4x: . Then I put the flask in the incubator at 37 degrees Celsius for 24 hours. Up next, changing the media.

7.13.12 Picogreen DNA assay and data analysis

Happy Friday! I apologize for leaving you hanging about the picogreen DNA assay results, but your wait is over:)

Monday I shared with you some of the background behind the picogreen DNA assay and how to read the results. So here’s what happened Tuesday:

I needed to run four different sets of samples in this experiment. I would be using light to measure how much DNA was present in a sample. I had my frozen day 1 and day 7 scaffolds of mouse and human bone marrow cells, a control of bacteriophage DNA that would show the buffer I used free the DNA worked properly, a dilution series of the same type of bacteriophage DNA to create the line of best fit, and a set of controls of buffer without DNA to make sure there was not any biological contaminates. That’s a lot right? Dr. Simon shared with me that much of the work he does involves test controls to make sure all components of an experiment are working properly.

Before I go on, I wanted to share an image of how a bacteriophage works: When I first learned about these amazing viruses I could not believe how cool they were in their appearance and delivery. Basically it is a virus that injects its DNA into bacteria so that the bacteria does the work of replicating the virus and assembling new phages. It looks and acts like something straight out a science fiction movie right?! That’s one of the many things I love about science, learning about this cool stuff! But, back to the assay…

My first task was to make the serial dilution. I would be diluting a concentration by a factor of 3 at each step with a total of 10 steps. I started with known concentration of 1000 ng/mL or 0.000001 g/mL (nano= 10-9). I placed 150 uL DNA sample into well 1. The I took 50 uL from well 1 and placed it into well 2 which already had 100 uL of buffer in the well. I then mixed up the contents in the well then transferred 50 uL from well 2 to well 3.  I continued this process 7 mores times. Each time I was careful to mix the contents before transferring the sample. Why? What would happen to the concentrations if I did not mix it? What might happen to the concentration if I had an air bubble in the tip of the pipette? Needless to say, this was a slow process.

Once the dilution series was complete, it was time to transfer 100uL of the DNA in lysis solution to this new plate
In order to make sure I knew where each sample would go I made a small mark on the first well on each row: I also marked the top of the plate with the location of each of my samples: and recorded this information in my lab book as well. Each time I transferred a sample I made sure the mix up the sample in the well and change the tip after moved the sample, Why? What was the point?

Next up I transferred the lysed bacteriophage DNA as one control and transferred a sample of plain buffer to 3 wells to run as another control. Now it was time to add the fluorescent tag o the DNA, the picogreen. This is a light-sensitive ingredient so I covered the vial with foil to reduce the amount of light entering the sample: and transferred 100 uL of picogreen in buffer to each of the wells: .

The it was time to run the samples in the fluorescence imaging machine: I loaded the plate onto the arm and then closed the drawer:  The machine is attached to a computer with specific software used to capture the data  as the plate is “read.” There is a mercury lightbulb similar to the one used to take the fluorescence images with the microscope: In addition there are numerous mirrors that beam the light through the sample. Picogreen has a specific excitation wavelength emits a certain wavelength (see 6-28-12-more-fluorescence-imaging-and-seeding-human-bone-marrow-stem-cells for more info on how this works) How bright this light is emitted  is called the fluorescence intensity and depends on how much of the tag is present. This tag binds to the DNA in our sample so the more tag, the more DNA!

It took about a minute to run the plate and here’s what the results looked like: We needed to export these results to an Excel spreadsheet in order to analyze them and determine if the experiment worked as it was supposed to and how much DNA was present on each scaffold. That’s what I did today.

I took the raw data created the standard curve with the line of best fit: and found the formula for the line of best fit in the y-intercept form. (See I told you math was important!!!!!)  I knew what the fluorescence intensity reading for each of my wells (y value), and using this formula, I could figure out what the concentration of DNA was in each well (x value). For example: the fluorescence intensity in well 1 was 11778. I plugged this y value into the formula: y = 1.8476x + 4.8972 where y is the value on the y axis, 1.8476 is the slope of the line (how far the line rises (change in y) as it runs (change is x)) and 4.8972 is where the line of best fit crosses the y-axis. If I lost you, check out Brainpop: Slope and Intercept (sligoms, brainpop). So when I substituted y in the formula with 1178= 1.8476x + 4.8972 I needed to solve for x. In order to do that I needed to subtract 4.8972 from both sides of the equation and I got 1173.1028 = 1.8476x Now I needed to divide both sides of the equation with 1.8476 to isolate x and I got 634. That meant I had 634 ng/mL of DNA in well 1.

But I still needed to know how much DNA was present without the volume since the cells were grown on the scaffold not in solution: . So I needed to figure out how to remove the mL units. Since I had worked with a sample of 200uL (0.2 uL) in each well, I could multiply the amount of DNA by 0.2 mL and that would cancel out the mL units. But I also needed to remember that my original lysed solution contained 200 uL and I only test 100 uL so I needed to multiply the amount of DNA by 2 to find the total amount of DNA on the scaffold. I used the formula DNA (ng) = 0.2 * d *  2 where d= the amount of DNA in solution. SO for well 1 I found DNA (ng) = 0.2 * 634 * 2 = 253 ng. I did this for all of my samples and created the following data tables:   In analyzing this data I found a few things. One, the mouse cells proliferated (reproduced) more easily than human cells. There were about twice as many cells on day 7 as on day 1. And something went very wrong with one of my scaffolds. Can you tell which one?  There was very little DNA on scaffold 3 in the day 7 from the hBMSC-8001 cells. Dr. Simon and I spent a long time discussing this piece of data and what to do with it. There are a number of statistical analysis tools scientists use to analyze their data and determine the chances of randomly getting their results. If one of the data points very different from the rest of the set, it is known as an outlier and we can do a statistical analysis to find the probability of getting that result again. If it is extremely unlikely to be reproducible ( how often I could repeat this test and get the same results) scientists can choose to remove the data point from their set. I was very hesitant about doing this. I wanted to make sure that I was being honest in my representation of the results as well as make sure I used this mistake as a learning experience ( try to think what went wrong so I don’t do it again). After much debating I agreed to create two representations of the data, one using scaffold 3 and one without it. Here’s the graphs of the results without the outlier. What do you notice about the average amount of DNA on day 7 of the hBMSC cells in the graph below compared to the amount in the graph above? You have to be careful to check the values of the y axis but you should see that the average amount of DNA in the graph above is close to 90 ng and the average amount of DNA in the graph below is close to 275 ng. One first glance, the graphs appear very similar, it is only with close study, you see the big difference in values. This is another way scientists may skew or misrepresent their results in what is known as showing a bias.

After taking a look at this data and the photographs taken in this experiment shown in this powerpoint  2012 Trial 1 hBMSC and mC3T3-E1, Dr. Simon and I agreed that I would perform one more experiment. I will repeat the same experiment I have done so far with one adjustment. We noticed that there were many more hBMSC cells that grew in the corners of the scaffolds than the mC3T3-E1 cells:  To determine if this was true we are going to add a third set of scaffolds to the experiment where we will grow both human and mouse osteoblasts for 14 days. At that time I will fix and stain and try to compare the amount of growth in the corner of the scaffolds. If this is true, it may lead into another experiment for Dr. Simon and his team. That’s the way research works. For every experiment, you analyze the data, compare and discuss the results, try to determine a theory as to why you saw the results you did and in many times you end up with more questions than you started with and this leads to your next project.

So up next? Growing mC3T3-E1 cells in culture on Monday!

7.9.12 Fluorescence Imaging and Picogreen Day 1

Good evening! I hope you are enjoying the “cooler” temperatures, what a difference 20 degrees makes! I was working on two tasks in the lab today, collecting reference scaffold images and preparing the frozen scaffolds for the Picogreen Assay.

This is a powerpoint showing mC3T3-E1 Fluorescence Imaging for days 1 and 7 on scaffold material. Each slide includes the ruler used to scale the images. This a powerpoint showing hBMSC-8001 Fluorescence imaging for days 1 and 7 on scaffold material. Again each slide includes the ruler used to scale the images. And this is the First trial of osteoblast cells on scaffold material. Each page contains 9 images, 3 from each scaffold, two top views, one at 4x and one at 10x, and one bottom view at 4x. The next step will be to draw some conclusions based on the observations of these photos. I will be presenting these results along with the Picogreen Assay results to the 3D scaffolding department later this month.

After finishing up collecting the photos it was time to prepare the scaffold samples for the Picogreen Assay. This is a test that measures the amount of DNA present in a sample using light. In order to be able the measure the DNA we have to add a fluorescent tag to it. But remember that DNA is coiled up in the nucleus. So my first step was to make a “detergent” that will break down proteins to free the DNA so that we can add the tag, called a “lysis buffer.” This buffer will break the cells open and degrade or break down the protein and free the DNA. After making this buffer solution I added 0.2 mLs to each well with the scaffold samples.

I also needed to make a control solution of dsDNA from a bacteriophage (ds= double stranded). A phage is like a virus that inserts it’s own DNA into bacteria. We used this DNA because it is relatively easy to obtain and inexpensive. This control solution will be used to create a serial dilution tomorrow. A serial dilution involves reducing a known concentration by a factor in a series. For example, a solution may start with a concentration of 1000 mg/mL and by taking a portion of the sample (say 10%) and adding liquid such as buffer or water (90%) you have reduced the concentration to 100mg/mL. You would continue to dilute the sample by adding more buffer to reduce the concentration further. For more on serial dilution, check out this video:

Since we will know the DNA concentration in each sample in the series we will be able to create graph of fluorescence intensity (y) (from the fluorescence tag) versus DNA concentration (x) and create a line of best fit like this:Then we will use this graph as a reference to compare the intensity of my DNA from each of the scaffolds to find the amount of DNA present on each scaffold. We then will use the concentration of DNA as a measure of how many cells are present of the scaffold. A very neat technique huh? Imagine trying to measure all of these cells: And this is only a section of the the top of the scaffold!

The assays that we completed in class only required us to observe the color changes of our sample and compare them to the controls like this: This assay will require a machine to determine the intensity of the fluorescent glow. The thought is the more DNA in a sample, the more fluorescent tag, and the greater the intensity. Based on the images we already collected what hypothesis would you make about the amount of DNA present on days 1 and 7? How about the amount of mouse DNA present compared to the amount of human DNA present? Check in tomorrow to see if your hypothesis was correct!

 

7.5.12 Merging and Coloring Images

Good afternoon! I hope you enjoyed the holiday and got to see some great fireworks. I wonder if you remembered to tell your family about how different elements give off different colors when their electrons jump energy levels. Need a refresher? Check out the post from Bohr Model and Flame Testing. If you want to find out more from about fireworks, check out Chemical of the Week: Fireworks. Isn’t science cool!?!?!!?!

My day in the lab involved learning how to merge and color the images of the cells on days 1 and 7. I was able to take the separate images taken of the same  location on the scaffold captured using the red filter to show the cytoskeleton of the mouse bone marrow cells: and using the green filter to show the nucleus of the same cells: and merged them into one image and added color to create this image: Again, how cool is that! Now I just need to add the scale and label the images. The plan for tomorrow is to fix and stain the samples for day 7 of the human bone marrow stem cells.