Talk:20.109(S13):Module 3

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==Protocols==
==Protocols==
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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.
+
<font color=purple>If you got to go to the TC room first on Day 2, you will go in the second cohort today (and vice-versa). If you are in the second group, use the time that you are waiting to complete your research idea discussion, and if you have time to also prepare your RNase-free area, label tubes that you will need, etc.</font color>
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===Part 1: Bead preparation for Live/Dead&reg; fluorescence assay===
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===Part 1: Research idea discussion===
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#Retrieve your 2 six-well dishes from the incubator.  
+
Before (second TC cohort) or after (first TC cohort) your wet-lab work today, take some time to discuss the five research results you wrote up for homework with your lab partner, guided by the instructions below.  
-
#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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#*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.
+
Writing a research proposal requires that you identify an interesting topic, spend lots of time learning about it, and then design some clever experiments to advance the field. It also requires that you articulate your ideas so any reader is convinced of your expertise, your creativity and the significance of your findings, should you have the opportunity to carry out the experiments you’ve proposed. To begin you must identify your research question. This may be the hardest part and the most fun. Fortunately you started by finding a handful of topics to share with your lab partner. Today you should discuss and evaluate the topics you’ve gathered. Consider them based on:
-
#*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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* your interest in the topic
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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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* the availability of good background information
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#Within the Petri dish, cut your whole beads in half using a spatula or razor blade.
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* your likelihood of successfully advancing current understanding
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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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* the possibility of advancing foundational technologies or finding practical applications
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#Per dish, rinse the beads with 3 mL of warm HEPES buffered saline solution (HBSS).
+
* if your proposal could be carried out in a reasonable amount of time and with non-infinite resources
-
#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.
+
 
-
#Incubate for 15 min. with the TC hood light off.
+
It might be that not one of the topics you’ve identified is really suitable, in which case you should find some new ideas. It’s also possible that through discussion with your lab partner, you’ve found something new to consider. Both of these outcomes are fine but by the end of today’s lab you should have settled on a general topic or two so you can begin the next step in your proposal writing, namely background reading and critical thinking about the topic. '''Check in with Thomas and get his feedback about your ideas for a few minutes before leaving today.''' 
-
#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.
+
 
-
#Rinse the cells with 3 mL HBSS buffer again. Pipet off as much liquid as possible, again into the Dye Collection tube.
+
A few ground rules that are 20.109 specific:
-
#Soak in 3 mL of 4% glutaraldehyde solution for 15 minutes.
+
*You should not propose any research question that has been the subject of your UROP or research experience outside of 20.109.  This proposal must be original. 
-
#Pipet off the solution, and then bring your Petri dish to the fluorescent microscope bench in the lab.
+
*You should keep in mind that this proposal will be presented to the class, so try to limit your scope to an idea that can be convincingly presented in a twelve minute oral presentation. 
-
#For observation, place the half-bead on a glass slide and then cover with a coverslip -- don't press down too hard.
+
 
-
#*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.
+
Once you and your partner have decided on a suitable research problem, it’s time to become an expert on the topic. This will mean searching the literature, talking with people, generating some ideas and critically evaluating them. To keep track of your efforts, you should start a wiki catalog on your OpenWetWare user page. How you format the page is up to you but check out the [[Yeast rebuild |“yeast rebuild”]] or the [[T7.2 | “T7.2”]]  wiki pages on OpenWetWare for examples of research ideas in process. As part of a later FNT assignment, you will have to print out your wiki page specifying your topic, your research goal and at least two helpful references that you’ve read and summarized.
-
#*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.
+
 
 +
===Part 2: Prepare cell lysates===
 +
 
 +
You will prepare cell-bead samples in three different ways: one will allow  you to count your cells, and is suitable for RNA preparation, while the other two will involve more stringent bead/matrix dissolution for better protein or proteoglycan recovery. Split up the work with your partner whatever way is most convenient. '''Remember to label your samples carefully at every step.'''
 +
 
 +
#Before proceeding, briefly observe the cell-bead constructs under the microscope and note any changes from Day 3.
 +
#*Let the teaching faculty know if you have difficulty focusing within a bead.
 +
#Remove the culture medium from each of your samples. Be careful not to suck up the beads; it will help to use a serological pipet just as you did when washing your freshly synthesized beads. Tipping the plate will help the beads settle in a cluster and allow you to remove medium elsewhere.
 +
#*A 5 mL pipet size should work well for rigid beads, while for more delicate beads, you should use a 2 mL serological pipet or even a P1000. If your beads are falling apart, you can transfer the beads according to steps 3-5 below without trying to remove medium first.
 +
#*If you are concerned about your bead amount, talk to the teaching faculty. You might skip the proteoglycan assay and focus on the other two instead.
 +
#About 1/3 of your beads will be used to measure protein content: move these to an eppendorf tube. The goal is about 10-15 (2-3 mm) beads per tube.
 +
#*For large beads (4-5 mm), you might use only 5-10 beads, and for very small beads (<1 mm), you might use 20 or more.  
 +
#Another 1/3 will be used to measure proteoglycan content; these whole beads can also be moved to an eppendorf tube.
 +
#The final 1/3 will be used to isolate RNA. Using a sterile spatula, transfer the beads into a fresh well of your 6-well plate. This transfer step is to exclude any cells that are growing on the bottom of the plate (as opposed to actually in the beads) from analysis.
 +
 
 +
====Samples for RNA Isolation====
 +
 
 +
#Rinse the transferred bead-cell constructs with 4 mL of warm PBS, then aspirate the buffer.
 +
#*If your beads are very fragile, you might want to skip the PBS rinse, and directly proceed to step 2.
 +
#Add 3 mL of pre-warmed EDTA-citrate buffer, and incubate at 37 &deg;C for 10 min.
 +
#*Meanwhile, prepare the beads for the protein and proteoglycan assays as described below. All the materials that you need are in eppendorf tubes in the fridge.
 +
#Now recover your cells:
 +
#*Add 3 mL of warm complete culture medium, pipet up and down to break up the beads (you may find this easier with a 1 mL pipetman rather than a serological pipet), and transfer to a 15 mL conical tube.  
 +
#*Spin the cells down at 1900g for 6 min (using the centrifuge that is in the TC room).  
 +
#Resuspend in ~ 1-1.5 mL of culture medium, and ''write down'' what you use. Mix thoroughly by pipetting, then set aside a 90 &mu;L aliquot of your cells for counting, and put the rest of the cells into another eppendorf tube.
 +
#*If you have very few cells based on your Day 3 observations and/or having very few beads, you might consider skipping the cell count, and instead keeping all of the cells for RNA isolation. If you have too few cells to get a reliable cell count, you are not losing valuable information for your report in any case. And if you have so few cells that taking some of them for a count compromises your other data, then that outcome would not be preferable to missing the cell count.  
 +
#While one of you begins the spin in the main lab (see Part 2), the other should count your cell aliquot as on Day 2, at a 9:1 ratio with Trypan blue. '''Separately''' calculate the approximate numbers of live (yellowish) and of dead (blue) cells.  
 +
#*Recall that you must multiply by 10,000 (and your dilution factor) to convert a hemacytometer cell count to a cells/mL concentration.
 +
 
 +
====Samples for Protein Extraction====
 +
 
 +
#Per eppendorf tube (typically 10-15 beads), add 133 &mu;L of cold EDTA-citrate buffer, and pipet up and down for 20-30 seconds to dissolve the beads. Be thorough while limiting bubbles as best you can. The resulting solution may be viscous.
 +
#Pipet 33 &mu;L of 0.25 M acetic acid into each eppendorf tube.
 +
#Finally, pipet 33 &mu;L of 1 mg/mL pepsin (in 50 mM acetic acid) into each tube and mix well.
 +
#Move your eppendorf tubes into the rack in the 4 &deg;C fridge. Tomorrow the teaching faculty will move them to an elastase solution (also at 4 &deg;C) to break down the polymeric collagen to more readily measured monomeric collagen.
 +
 
 +
====Samples for Proteoglycan Extraction====
 +
 
 +
#Soak your beads for a few minutes in pre-warmed PBS, and then remove as much of the PBS as possible. Shoot to have '''no''' pink tint to the beads, as it is known to interfere with the proteoglycan assay.
 +
#Add 250 &mu;L of papain solution to your beads. The papain is in an EDTA-citrate buffer base.
 +
#When the first partner goes to the main lab, s/he should take this tube to the 60 &deg;C heat block. After 24 hours, the samples will be moved to the fridge.

Current revision

Contents

Protocols

If you got to go to the TC room first on Day 2, you will go in the second cohort today (and vice-versa). If you are in the second group, use the time that you are waiting to complete your research idea discussion, and if you have time to also prepare your RNase-free area, label tubes that you will need, etc.

Part 1: Research idea discussion

Before (second TC cohort) or after (first TC cohort) your wet-lab work today, take some time to discuss the five research results you wrote up for homework with your lab partner, guided by the instructions below.

Writing a research proposal requires that you identify an interesting topic, spend lots of time learning about it, and then design some clever experiments to advance the field. It also requires that you articulate your ideas so any reader is convinced of your expertise, your creativity and the significance of your findings, should you have the opportunity to carry out the experiments you’ve proposed. To begin you must identify your research question. This may be the hardest part and the most fun. Fortunately you started by finding a handful of topics to share with your lab partner. Today you should discuss and evaluate the topics you’ve gathered. Consider them based on:

  • your interest in the topic
  • the availability of good background information
  • your likelihood of successfully advancing current understanding
  • the possibility of advancing foundational technologies or finding practical applications
  • if your proposal could be carried out in a reasonable amount of time and with non-infinite resources

It might be that not one of the topics you’ve identified is really suitable, in which case you should find some new ideas. It’s also possible that through discussion with your lab partner, you’ve found something new to consider. Both of these outcomes are fine but by the end of today’s lab you should have settled on a general topic or two so you can begin the next step in your proposal writing, namely background reading and critical thinking about the topic. Check in with Thomas and get his feedback about your ideas for a few minutes before leaving today.

A few ground rules that are 20.109 specific:

  • You should not propose any research question that has been the subject of your UROP or research experience outside of 20.109. This proposal must be original.
  • You should keep in mind that this proposal will be presented to the class, so try to limit your scope to an idea that can be convincingly presented in a twelve minute oral presentation.

Once you and your partner have decided on a suitable research problem, it’s time to become an expert on the topic. This will mean searching the literature, talking with people, generating some ideas and critically evaluating them. To keep track of your efforts, you should start a wiki catalog on your OpenWetWare user page. How you format the page is up to you but check out the “yeast rebuild” or the “T7.2” wiki pages on OpenWetWare for examples of research ideas in process. As part of a later FNT assignment, you will have to print out your wiki page specifying your topic, your research goal and at least two helpful references that you’ve read and summarized.

Part 2: Prepare cell lysates

You will prepare cell-bead samples in three different ways: one will allow you to count your cells, and is suitable for RNA preparation, while the other two will involve more stringent bead/matrix dissolution for better protein or proteoglycan recovery. Split up the work with your partner whatever way is most convenient. Remember to label your samples carefully at every step.

  1. Before proceeding, briefly observe the cell-bead constructs under the microscope and note any changes from Day 3.
    • Let the teaching faculty know if you have difficulty focusing within a bead.
  2. Remove the culture medium from each of your samples. Be careful not to suck up the beads; it will help to use a serological pipet just as you did when washing your freshly synthesized beads. Tipping the plate will help the beads settle in a cluster and allow you to remove medium elsewhere.
    • A 5 mL pipet size should work well for rigid beads, while for more delicate beads, you should use a 2 mL serological pipet or even a P1000. If your beads are falling apart, you can transfer the beads according to steps 3-5 below without trying to remove medium first.
    • If you are concerned about your bead amount, talk to the teaching faculty. You might skip the proteoglycan assay and focus on the other two instead.
  3. About 1/3 of your beads will be used to measure protein content: move these to an eppendorf tube. The goal is about 10-15 (2-3 mm) beads per tube.
    • For large beads (4-5 mm), you might use only 5-10 beads, and for very small beads (<1 mm), you might use 20 or more.
  4. Another 1/3 will be used to measure proteoglycan content; these whole beads can also be moved to an eppendorf tube.
  5. The final 1/3 will be used to isolate RNA. Using a sterile spatula, transfer the beads into a fresh well of your 6-well plate. This transfer step is to exclude any cells that are growing on the bottom of the plate (as opposed to actually in the beads) from analysis.

Samples for RNA Isolation

  1. Rinse the transferred bead-cell constructs with 4 mL of warm PBS, then aspirate the buffer.
    • If your beads are very fragile, you might want to skip the PBS rinse, and directly proceed to step 2.
  2. Add 3 mL of pre-warmed EDTA-citrate buffer, and incubate at 37 °C for 10 min.
    • Meanwhile, prepare the beads for the protein and proteoglycan assays as described below. All the materials that you need are in eppendorf tubes in the fridge.
  3. Now recover your cells:
    • Add 3 mL of warm complete culture medium, pipet up and down to break up the beads (you may find this easier with a 1 mL pipetman rather than a serological pipet), and transfer to a 15 mL conical tube.
    • Spin the cells down at 1900g for 6 min (using the centrifuge that is in the TC room).
  4. Resuspend in ~ 1-1.5 mL of culture medium, and write down what you use. Mix thoroughly by pipetting, then set aside a 90 μL aliquot of your cells for counting, and put the rest of the cells into another eppendorf tube.
    • If you have very few cells based on your Day 3 observations and/or having very few beads, you might consider skipping the cell count, and instead keeping all of the cells for RNA isolation. If you have too few cells to get a reliable cell count, you are not losing valuable information for your report in any case. And if you have so few cells that taking some of them for a count compromises your other data, then that outcome would not be preferable to missing the cell count.
  5. While one of you begins the spin in the main lab (see Part 2), the other should count your cell aliquot as on Day 2, at a 9:1 ratio with Trypan blue. Separately calculate the approximate numbers of live (yellowish) and of dead (blue) cells.
    • Recall that you must multiply by 10,000 (and your dilution factor) to convert a hemacytometer cell count to a cells/mL concentration.

Samples for Protein Extraction

  1. Per eppendorf tube (typically 10-15 beads), add 133 μL of cold EDTA-citrate buffer, and pipet up and down for 20-30 seconds to dissolve the beads. Be thorough while limiting bubbles as best you can. The resulting solution may be viscous.
  2. Pipet 33 μL of 0.25 M acetic acid into each eppendorf tube.
  3. Finally, pipet 33 μL of 1 mg/mL pepsin (in 50 mM acetic acid) into each tube and mix well.
  4. Move your eppendorf tubes into the rack in the 4 °C fridge. Tomorrow the teaching faculty will move them to an elastase solution (also at 4 °C) to break down the polymeric collagen to more readily measured monomeric collagen.

Samples for Proteoglycan Extraction

  1. Soak your beads for a few minutes in pre-warmed PBS, and then remove as much of the PBS as possible. Shoot to have no pink tint to the beads, as it is known to interfere with the proteoglycan assay.
  2. Add 250 μL of papain solution to your beads. The papain is in an EDTA-citrate buffer base.
  3. When the first partner goes to the main lab, s/he should take this tube to the 60 °C heat block. After 24 hours, the samples will be moved to the fridge.
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