Talk:20.109(S13):Module 3: Difference between revisions

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(New page: ==Protocols== Half the class at a time will work in the tissue culture room today. Today will be physically and mentally laborious, and you've all been working hard, so spend the rest of ...)
 
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==Protocols==
===Part 2: Microscopy===


Half the class at a time will work in the tissue culture room today. Today will be physically and mentally laborious, and you've all been working hard, so spend the rest of the afternoon however you see fit. (Whether that involves the FNT assignment, notebook prep, or a walk in the sunshine - are we still expecting sunshine?)
When observing your cells under fluorescence excitation, you should work with the room lights off for best results. A member of the teaching faculty will be with you to help you make the most of your 20-25 minutes.


===Chondrocyte or stem cell culture===
#Prior to the first group using the microscope, the teaching faculty will turn on the microscope and allow it to warm up for 15-20 min. First, on the mercury lamp that is next to the microscope, the ‘POWER’ switch will be flipped. Next, the ‘Ignition’ button will be held down for about a second, then released.  
 
#When you arrive, the ''lamp ready'' and ''power'' indicators should both be lit – talk to the teaching faculty if this is not the case.  
Today you will work with primary cells that are directly isolated from bovine knee joints. Recently, your teaching faculty harvested cartilage fragments from two bovine knees, and sequentially digested them in pronase and collagenase enzymes to release the chondrocytes. Each joint typically yields > 50-100M cells. Stem cells were harvested from the bone marrow and grown up from a rare population by extended culture in bFGF (basic fibroblast growth factor). After cell isolation, aliquots of several million cells each were frozen and stored in liquid nitrogen.  
#Place your first sample slide on the microscope, coverslip-side up, by pulling away the left side of the metal sample holder for a moment.
 
#Begin your observations with the 10X objective.  
====Preparation====
[[Image:20109_fluor-scope_front.jpg|thumb|left|250px|Fluorescent microscope, front view.]]
 
[[Image:20109_fluor-scope_side.jpg|thumb|center|250px|Fluorescent microscope, side view.]]
#Begin by setting up your hoods. Prepare any standard equipment and solutions needed.
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#Note that the small beakers are for making a calcium chloride bath (not shared, one per person), and the large are for temporary waste in steps 10-12 below (shared, one per hood).
#Turn on the illumination using the button at the bottom left of the microscope body (on the right-hand side is a light intensity slider).  
#If you requested a special reagent or equipment, check with the teaching faculty.
#Next, turn the excitation light slider at the top of the microscope to ‘DIA-ILL’ (position 4).
#If you are doing an alternative protocol (e.g., 2D culture or collagen gels), check with the teaching faculty.
#Try to focus your sample. However, be aware that the contrast is not great for your cells, and you might not be able to focus unless you find a piece of debris. Whether or not you find focus, after a minute or two, switch over to fluorescence. Your cells will be easier to find this way.
 
#*First, turn the white light illumination off.
====Cell culture====
#*Next, move the excitation slider to ‘FITC’ (position 3). You should see a blue light coming from the bottom part of the microscope.  
 
#**This light can excite both the green and the red dye in the viability kit, and the associated filter allows you to view both colors at once.  
#When your hood is ready, thaw your aliquot(s) of frozen cells in the water bath. Avoid immersing the cap of the tube in the bath; just hold the body submerged.  Agitate the vial slightly while you hold it. The cells should thaw in less than 5 minutes.
#*Finally, you must slide the light shield (labeled ‘SHUTTER’) to the right to unblock it. Now you can look in the microscope, and use the coarse focus to find your cells (which should primarily be bright green), then the fine focus to get a clearer view.
#Spray the vial with 70% ethanol and take it into your hood. Using a P1000, add the cells drop-wise into the 15 mL conical containing 9 mL of pre-warmed medium. Spin at 800 g for 8 minutes.  
#*You can also switch the excitation slider over to ‘EthD-1’ (position 2) to see only the red-stained cells. Some of your cells may appear to be dimly red, but the dead ones are usually obviously/brightly stained.
#Aspirate most of the medium off your cell pellet, then gently resuspend in 1 mL of medium using your P1000. Add 3 mL more of medium per vial, using a serological pipet for the addition and subsequent mixing of the medium and cells. Take 90 &mu;L of cells into an eppendorf tube.
#*'''Be aware that the dyes do fade upon prolonged exposure to the excitation light, so don’t stay in one place too long, and when you are not actively looking in the microscope, slide the light shield back into place.'''
#Add 10 &mu;L of Trypan blue - '''this is a toxic material, so please be careful not to spill it!''' - to the eppendorf tube, and count your cells. Adjust your culture plan if you do not have as many cells as you expected.  
#You can try looking at your cells with the 40X objective as well if you have time. As you move between objectives and samples, choose a few representative fields to take pictures of. '''As a minimal data set, try to get 3 fields at 10X of both of your samples.'''
#*No need to count all 4 corners today - perhaps count 2, especially if your cell count is high.
#*To take a picture, remove one eyepiece from the microscope, and replace it with the camera adaptor. Be sure to keep the light shield in place until you are ready to take the picture (to avoid photobleaching)!
#Separate the cells that will make up your two different cultures into two labeled 15 mL conical tubes. Note that the tubes may not all require the same amount of cells, depending on the cell densities you chose for the two cultures. Double-checking your calculations now may save you having to do an extra centrifugation step later!
#*Note that 10X images will reveal a broader field, but 40X images may have better contrast.  
#*Give any excess cells that you have to the teaching faculty, in case other groups want more cells.
#*Check with the teaching faculty if you are having difficulty getting clear pictures.
#Spin down your two conical tubes of cells at 800 g for 8 minutes.
#*Later in the module, you will compare the average cell numbers in each sample using the statistical methods we discussed during Module 2.
#Resuspend each sample of cells in the appropriate amount of the type and concentration of alginate that you chose. 
#Post two well-captioned pictures to the wiki before leaving (one of each sample), so we can discuss the class data in our next lecture. Be sure to note whether the image is at the surface or core of the bead.
#Using the syringe that has been prepared for you, very carefully pull up the cells, then release them drop-by-drop into the beaker full of calcium chloride solution (20 mL). Recall that calcium effectively polymerizes the alginate, resulting in small gel beads filled with cells. '''Immediately discard the entire syringe into the RED sharps container (not the mayo jars) - do not try to remove or recap the needle.'''  
#*If you are one of the last two groups to use the microscope, you may post your data within 24 hours instead.
#*Don't release too quickly or you will get a glob instead of distinct droplets, and try to match your release rate with your partner's.
#*Depending on the concentration of alginate that you chose, you may have between ~50-150 beads for 1 mL of alginate solution.
#Allow the polymerization to proceed for 10 min. at room temperature. Then pour your beads into a 50mL conical tube.  
#Remove the calcium chloride solution from your beads using a large serological pipet (to better avoid aspirating the beads), and put this solution in the temporary waste beaker in your hood.  
#*Ask the teaching faculty for tips on avoiding sucking up your beads. Basically, you want to keep the pipet close to the wall of the conical tube, so liquid can still be sucked up but the beads don't have room to be.
#Now fill the conical tube with sodium chloride (20 mL), and gently invert it for 1-2 min. This is to remove excess calcium from the solution.
#Remove the NaCl using a fresh pipet, then wash the beads again with fresh NaCl. Finally, wash the beads two times with DMEM culture medium (20 mL each time).
#For each of your two samples, transfer the beads to the two leftmost wells of a 6-well plate, using a sterile spatula. Try to put approximately equal numbers of beads in the two wells.
#Finally, add 6 mL of warm culture medium to each of your four sample wells, then put the two well-plates in the incubator.
 
The teaching faculty will exchange the culture medium as necessary (every other day).

Revision as of 07:36, 25 April 2013

Part 2: Microscopy

When observing your cells under fluorescence excitation, you should work with the room lights off for best results. A member of the teaching faculty will be with you to help you make the most of your 20-25 minutes.

  1. Prior to the first group using the microscope, the teaching faculty will turn on the microscope and allow it to warm up for 15-20 min. First, on the mercury lamp that is next to the microscope, the ‘POWER’ switch will be flipped. Next, the ‘Ignition’ button will be held down for about a second, then released.
  2. When you arrive, the lamp ready and power indicators should both be lit – talk to the teaching faculty if this is not the case.
  3. Place your first sample slide on the microscope, coverslip-side up, by pulling away the left side of the metal sample holder for a moment.
  4. Begin your observations with the 10X objective.
Fluorescent microscope, front view.
Fluorescent microscope, side view.


  1. Turn on the illumination using the button at the bottom left of the microscope body (on the right-hand side is a light intensity slider).
  2. Next, turn the excitation light slider at the top of the microscope to ‘DIA-ILL’ (position 4).
  3. Try to focus your sample. However, be aware that the contrast is not great for your cells, and you might not be able to focus unless you find a piece of debris. Whether or not you find focus, after a minute or two, switch over to fluorescence. Your cells will be easier to find this way.
    • First, turn the white light illumination off.
    • Next, move the excitation slider to ‘FITC’ (position 3). You should see a blue light coming from the bottom part of the microscope.
      • This light can excite both the green and the red dye in the viability kit, and the associated filter allows you to view both colors at once.
    • Finally, you must slide the light shield (labeled ‘SHUTTER’) to the right to unblock it. Now you can look in the microscope, and use the coarse focus to find your cells (which should primarily be bright green), then the fine focus to get a clearer view.
    • You can also switch the excitation slider over to ‘EthD-1’ (position 2) to see only the red-stained cells. Some of your cells may appear to be dimly red, but the dead ones are usually obviously/brightly stained.
    • Be aware that the dyes do fade upon prolonged exposure to the excitation light, so don’t stay in one place too long, and when you are not actively looking in the microscope, slide the light shield back into place.
  4. You can try looking at your cells with the 40X objective as well if you have time. As you move between objectives and samples, choose a few representative fields to take pictures of. As a minimal data set, try to get 3 fields at 10X of both of your samples.
    • To take a picture, remove one eyepiece from the microscope, and replace it with the camera adaptor. Be sure to keep the light shield in place until you are ready to take the picture (to avoid photobleaching)!
    • Note that 10X images will reveal a broader field, but 40X images may have better contrast.
    • Check with the teaching faculty if you are having difficulty getting clear pictures.
    • Later in the module, you will compare the average cell numbers in each sample using the statistical methods we discussed during Module 2.
  5. Post two well-captioned pictures to the wiki before leaving (one of each sample), so we can discuss the class data in our next lecture. Be sure to note whether the image is at the surface or core of the bead.
    • If you are one of the last two groups to use the microscope, you may post your data within 24 hours instead.
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