Talk:20.109(S13):Module 3

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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==
==Protocols==
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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?)
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<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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===Chondrocyte or stem cell culture===
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===Part 1: Research idea discussion===
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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.  
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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.  
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====Preparation====
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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
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#Begin by setting up your hoods. Prepare any standard equipment and solutions needed.
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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.''' 
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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).
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#If you requested a special reagent or equipment, check with the teaching faculty.
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#If you are doing an alternative protocol (e.g., 2D culture or collagen gels), check with the teaching faculty.
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====Cell culture====
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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. 
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#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.
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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.
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#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.
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#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.
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#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.
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#*No need to count all 4 corners today - perhaps count 2, especially if your cell count is high.
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#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!
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#*Give any excess cells that you have to the teaching faculty, in case other groups want more cells.
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#Spin down your two conical tubes of cells at 800 g for 8 minutes.
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#Resuspend each sample of cells in the appropriate amount of the type and concentration of alginate that you chose.  
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#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.'''
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#*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.
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#*Depending on the concentration of alginate that you chose, you may have between ~50-150 beads for 1 mL of alginate solution.
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#Allow the polymerization to proceed for 10 min. at room temperature. Then pour your beads into a 50mL conical tube.
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#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.
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#*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.
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#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.
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#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).
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#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.
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#Finally, add 6 mL of warm culture medium to each of your four sample wells, then put the two well-plates in the incubator.
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The teaching faculty will exchange the culture medium as necessary (every other day).
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===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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