Talk:20.109(S13):Module 3

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==Protocols==
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===Part 2: Microscopy===
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Today you can stagger your arrivals to lab (see today’s [[Talk:20.109%28S13%29:Testing_cell_viability_%28Day3%29 | Talk]] page). Only one group at a time will be able to work on the microscope, and assuming that cell culture setup takes ~ 1 hour, you will each have ~20-25 minutes to spend on the microscope. '''Please be respectful of your labmates’ time.''' Reading the protocol in advance will help you work more quickly, and is strongly recommended.
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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.
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===Part 1: Bead preparation for Live/Dead® fluorescence assay===
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#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.  
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#When you arrive, the ''lamp ready'' and ''power'' indicators should both be lit – talk to the teaching faculty if this is not the case.
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#Retrieve your 2 six-well dishes from the incubator.
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#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. 
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#The teaching faculty counted your beads during a recent media exchange (they are easiest to count in the absence of media). Based on the numbers written on your plate, decide how many beads (1-3 per sample) you can spare for today's assay. Ideally, for the three assays on Day 4 you want at least 45-60 beads total remaining (perhaps 30 or fewer for large beads). Be sure to take your bead(s) from only one of the two wells, just in case you contaminate it.  
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#Begin your observations with the 10X objective.  
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#*Also take this time to describe bead uniformity in your notebook, as this feature may affect your eventual experimental outcomes. Some groups had more luck than others in keeping bead size consistent between and within their two samples.
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[[Image:20109_fluor-scope_front.jpg|thumb|left|250px|Fluorescent microscope, front view.]]
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#*During a later incubation step, you might also take a look at your plate under the microscope, and focus in on cells within the beads. What is cell morphology and density like in each sample? Are there any cells growing ''under'' the beads, as a monolayer on the surface of the plate? Keep in mind that these will compete for media nutrients with the cells inside the beads.
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[[Image:20109_fluor-scope_side.jpg|thumb|center|250px|Fluorescent microscope, side view.]]
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#Using a sterile spatula, remove the beads (keeping the two samples separate) to two labeled Petri dishes. Do your best to keep the beads remaining in the culture wells sterile – the cells have to stay alive for 5 more days. Briefly dip the sterile spatula into the well, and immediately return your plate to the incubator, onto the shelf from which you took it.
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#Within the Petri dish, cut your whole beads in half using a spatula or razor blade.
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#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).  
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#*Small beads may be difficult to cut in half – if so, look at the intact bead instead.
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#Next, turn the excitation light slider at the top of the microscope to ‘DIA-ILL’ (position 4).
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#Per dish, rinse the beads with 3 mL of warm HEPES buffered saline solution (HBSS).
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#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.
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#Aspirate the HBSS - this may be easiest/safest to do with a P1000 - then pipet 200 &mu;L of dye solution right on the beads.
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#*First, turn the white light illumination off.
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#Incubate for 15 min. with the TC hood light off.
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#*Next, move the excitation slider to ‘FITC’ (position 3). You should see a blue light coming from the bottom part of the microscope.  
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#Remove the entire supernatant with a pipet, and expel it in the conical tube labeled ''Dye Collection''. The dye waste will be disposed of by the teaching faculty. You should also throw the pipet tip into the container on the microscope bench; tips will later be disposed of as solid waste in the chemical fume hood. You do ''not'' need to throw any later tips away here, as the dye will then be very dilute.
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#**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.  
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#Rinse the cells with 3 mL HBSS buffer again. Pipet off as much liquid as possible, again into the Dye Collection tube.
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#*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.
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#Soak in 3 mL of 4% glutaraldehyde solution for 15 minutes.  
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#*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.
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#Pipet off the solution, and then bring your Petri dish to the fluorescent microscope bench in the lab.
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#*'''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.'''
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#For observation, place the half-bead on a glass slide and then cover with a coverslip -- don't press down too hard.
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#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.'''
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#*You will probably want to look at the beads both flat side up (to see the core) and flat side down (to see the surface), time permitting.
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#*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)!
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#*You can make a "map" of the beads in your notebook and/or on the white surface of the slide. For example, you might have one bead on the left that is core side up and another on the right that is surface side up.
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#*Note that 10X images will reveal a broader field, but 40X images may have better contrast.
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#*Check with the teaching faculty if you are having difficulty getting clear pictures.
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#*Later in the module, you will compare the average cell numbers in each sample using the statistical methods we discussed during Module 2.
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#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.
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#*If you are one of the last two groups to use the microscope, you may post your data within 24 hours instead.

Revision as of 09:36, 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, front view.
Fluorescent microscope, side 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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