20.109(S10): TA notes for module 3

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General notes

Scheme: Each pair of students will make two cultures, with various modifications as proposed on Day 1.

The TA notes below are complemented by a Google Doc for buffer and aliquot calculations.

Key preparation:

Cell derivation from bovine knee joints (from Research 87, Inc.)

  • Please contact Agi Stachowiak by email for full derivation protocols (internal to Grodzinsky lab)
  • Chondrocytes
    • 2-day process (1 afternoon and 1 morning), then can be frozen away
  • Mesenchymal stem cells (MSCs)
    • initially a 2-day process to get to plating
    • 5-7 days to grow out (can be frozen at that time temporarily)
    • then expand for 2 passages over a few days each time, freeze
  • Initial time-intensive part should be done on a non-lab day (Spring Break or President's Day)

Lots of media to prepare. Some components go bad quickly (proline and ascorbate) and should be added on a daily basis to small amounts of media. Other components (pen/strep/amph, non-essential amino acids, etc.) can be added to a full DMEM bottle to make a base medium.

Note to instructors interested in implementing this lab:

Several changes are being piloted in the summer of 2010 to make the lab more technically robust:

  • Improve ELISA signal
    • completed - can email me for details
    • in short: after the one day of pepsin digestion, one day of elastase digestion should be done in TBS buffer, pH 8.0
  • Add a proteoglycan (PG) assay
    • completed - can email me for details
    • the standard DMMB assay must be modified for alginate cultures
    • at very low pH, sulfated PG but not alginate are recognized by the DMMB dye
    • based on Enobakhare, et al. in Analytical Biochemistry 243, 189-191 (1996)
    • took similar amount of beads as for collagen assay in a 250 μL volume (of papain/digestion buffer) for a good signal
  • Implement qPCR instead of semi-quantitative gel method
    • in progress - can email me for updates

Daily Notes

Day 1

No Quiz

Materials required:

  1. None: all work today is computer work. Get passing familiarity with the faculty-selected journal articles.

Day 2

Materials required:

  • Make sure to go through each group's plan to know which factors will be modified.
  1. 150 mM NaCl in autoclaved water--needs to be sterile filtered
  2. 102 mN CaCl2 in autoclaved water--needs to be sterile filtered
  3. Cell culture media
  4. Alginate--needs to be made the day before lab, allowed to dissolve at 4 °C overnight, then sterile filtered

Day of Lab:

  • Quiz (prepared by TA)
  • Sterile-filter all alginate on Thursday morning!
  • Media already has NEAA, HEPES, PSA; need to add proline, ascorbate, and FCS/ITS
  • To be autoclaved, per group
    • 2 beakers for CaCl2 bath
    • 1 sterile spatula, plus couple of extra
  • To be aliquoted, per group unless stated otherwise
    • Per microscope, 50 μL aliquot of Trypan blue in eppendorf tube
    • 15 mL conical with exactly 9 mL of medium (per group, plus couple of extra)
    • Other media: 4 mL + 4x 20 mL washes + 24 mL of final version = ~125 mL for 15% excess
    • Have the 24 mL final media in a separate tube, warmed up a bit later, kept cleaner
    • (2) 20ml aliquots of CaCl2
    • (4) 20ml aliquots of NaCl per group; bottles with 185 mL should be good per 2 groups
    • 2ml alginate
  • To be set up per hood ahead of time
    • Aspirators set up and tested
    • 2 pipet aids
    • 2 beakers with CaCl2 (pre-warmed); 2 for second group kept in teaching hood
    • 1 eppendorf tube for counting
    • both sizes of tips and pipetmen, set on 1 mL and 90 μL, respectively
  • To be available (dry/equipment), per group unless stated otherwise
    • 2 sterile 1 mL syringes and 21 G needles
    • 2 six-well plates
    • Bunch of 25 mL pipets

Spring 2010 specific:

  • T/R media needs
    • 6 of 7 groups will just get regular medium, but some will add additives
    • 1 of 7 groups will get regular medium for one sample, and low-FCS/ITS medium for the other
    • thus, total CDR medium (20% excess) needed is ~ 900 mL
    • total special medium needed is ~ 130 mL
  • T/R other needs
    • 1 group adding EDTA (release buffer, really) at 1:50
    • 1 group adding triple proline, means 96 μL per 6 mL
    • 1 group adding double ascorbate, means 3 μL per 6 mL
    • 2 groups playing with pH: will need to pre-test HEPES and NaHCO3 in main lab during first half
    • 1 group doing mechanical compression, will need to figure out set-up during first half (for now, sterilize some slides and weights)
  • W/F media needs
    • 5 of 7 groups will just get regular medium, but some will add additives
    • 1 of 7 groups will get regular medium for one sample, and low-FCS/ITS medium for the other
    • 1 of 7 groups will get stem cell medium
    • thus, total CDR medium (15% excess) needed is ~ 790 mL
    • total special medium needed is ~ 65 mL
    • total stem cell medium needed is ~ 135 mL
  • W/F other needs
    • 1 group adding bFGF at 15 ng/mL, or 0.6 μL per 12 mL, or 6 μL of a 1:10 dilution
    • 1 group is using 51 mM and 204 mM CaCl2, instead of 102 mM - need to prep and filter > 20 mL of each
    • 1 group is using a vortex on one plate - clean and put in separate incubator from rest
    • 1 group gets chondroitin sulfate - need to prepare appropriate stock
  • Media exchanges over course of module
    • Should be 3x for each group with all wells, then 2x more with half wells remaining
    • Therefore, per section will need approx. 700 mL media more for semester, or ~ 1.5 L on top of the ~ 1 L needed on the first day

Day 3

Materials required:

  1. HBSS (recipe below)
  2. dye solution in HBSS
  3. 4% glutaraldehyde (GAH) in HBSS
    • Prepared that day from 50% GAH stock
  4. 1 sterile spatula per group
  5. 1 50ml conical tube for waste per hood
  6. 2 Petri dishes per group
  7. slides and coverslips
  8. Camera and memory disks out

Day of Lab:

  • No Quiz
  • TA will stay with students in TC as they prepare their beads
  • Instructor will stay in the main lab and train students on microscopy

Day 4

Materials required:

  • Cell prep
    • lots of empty eppendorfs (don't need to be sterile)
    • waste tubes or beakers, for aspirations with ser. pipets
    • EDTA-citrate buffer (6 mL per group)
    • complete(ish) medium (9 mL per group)
    • a few eppendorfs w/100 μL of Trypan aliquotted
    • 4 eppendorfs of each of the following per day: 0.6 mL EDTA-citrate, 0.2 mL acetic acid, 0.2 mL pepsin
  • RNA prep and RT-PCR
    • 5 ice buckets
    • Water aliquots for spec. measurement
    • Thaw RT-PCR reagents (esp. water) early
    • RLT + β-merc last minute
    • Prep Master Mixes last minute
    • Turn on spec. last minute

Day of Lab:

  • Quiz
  • See "last minute" above
  • Note that g = rcf

Day 5

Materials required:

  • ELISA Day 1
    • (2) 96-well plates per group
    • (1) 300ul aliquot of CN I standard per group
      • prep 3.5 mL plus 35 μL
    • (1) 300ul aliquot of CN II standard per group
      • prep 3.5 mL plus 35 μL
    • PBS for diluting standards
    • eppendorfs
    • wash buffer
    • block buffer
    • primary antibodies at 1:4000
      • total needed per antibody ~ 25 mL
      • 3 batches of 10 mL PBS + 2.5 μL antibody
    • parafilm
  • Agarose gel
    • loading dye
    • sterile water
    • (2) 1.2% gels per day

Day of Lab:

  • Quiz
  • RT-PCR products out on cold block
  • protein samples briefly thawed just before lab

Day 6

Materials required:

  • ELISA Day 2
    • secondary antibody (1:1000, prep 10-15 mL at a time, in block buffer)
    • wash buffer
    • development buffer (1ml of development in 4ml of water per group)
    • pNPP (p-nitrophenyl phosphate) pellets (1 per 5 mL buffer, i.e., per group)
    • aluminum foil
    • stop solution (0.4 M NaOH)

Day of Lab:

  • Quiz
    • couple of multichannels at the sink, couple up front
    • students add antibody, development solutions at front bench, sharing 2 dishes per soln.

Day 7

Materials required: None--all computer work today

Day of Lab:

  • Quiz (final one)

Special materials

  • Chondrocyte Growth medium
    • Hi-glucose DMEM
    • 10% FCS (or 0.2% FCS with ITS, for more defined media)
    • Penicillin/Streptomycin/Amphotericin B
      • 100X antibiotic/antimycotic from Sigma
    • Non-essential amino acids
    • Sodium pyruvate
    • Proline (400 μM)
      • Stock: 11.5 mg/mL in DMEM, freeze single-use aliquots
    • HEPES (10 mM)
    • Ascorbate(20 μg/mL)
      • Stock: 20 mg/mL in water, sterile-filter, aliquot and freeze
  • Stem Cell Differentiation Medium
    • Hi-glucose DMEM
    • FCS and/or ITS+1 (insulin/transferrin/selenium)
    • Penicillin/Streptomycin/Amphotericin B
    • Non-essential amino acids
    • Sodium pyruvate
    • Proline (400 μM)
    • HEPES (10 mM)
    • Chondrogenic factors
      • TGF-beta1 (10 ng/mL)
      • Dexamethasone (100 nM)
      • Ascorbate (40 μg/mL)
  • Stem Cell Expansion Medium
    • Low-glucose DMEM
    • 10% FCS
    • Penicillin/Streptomycin/Amphotericin B
    • HEPES buffer, 10 mM (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)
    • up to 5 ng/mL bFGF (basic fibroblast growth factor)
  • Release Buffer (weights shown for 0.5 L)
    • 55 mM sodium citrate (8.09 g)
    • 30 mM EDTA (5.58 g)
    • 0.15 M NaCl (4.38 g)
    • pH to 6.8
    • sterile filter
  • HEPES buffer
    • stock is 1 M HEPES
    • leave excess volume for adding base, don't just add water all the way
    • pH to 7.2 (for 100 mL total, initially try 0.5 mL of 1 M NaOH - always test pH and add more as needed)
    • sterile filter
  • HEPES-buffered saline solution (HBSS)
    • 135 mM NaCl
    • 5 mM KCl
    • 1 mM MgSO4
    • 1.8 mM CaCl2
    • 10 mM HEPES
    • pH to 7.4
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