20.109(S11): TA notes for module 3
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.
Update to reflect pilots done this summer:
- Improve ELISA signal
- 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
- 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
Daily Notes
Day 1
No Quiz
Materials required:
- 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.
- 150 mM NaCl in autoclaved water--needs to be sterile filtered
- 102 mN CaCl2 in autoclaved water--needs to be sterile filtered
- Cell culture media
- 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!
- Prepare base media with NEAA, HEPES, PSA; then add proline, ascorbate, and FCS/ITS as needed
- To be autoclaved, per group
- 2 beakers for CaCl2 bath
- 1 sterile spatula, plus couple of extra
- To be aliquotted, 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 2011 specific:
- T/R media needs
- X of 7 groups will just get regular medium, but some will add additives
- thus, total CDR medium (20% excess) needed is ~ X mL
- total special medium needed is ~ X mL
- T/R other needs
- Red, Yellow, and Orange are using 20 and 200mM CaCl2 instead of the default
- Green, Blue, and Purple need alginate with HA at 0.5 and 2.5 mg/mL, and also CNII available
- We may need to make and sterile-filter the HA separately at 100X stock, because we won't have it until Thursday and then the alginate may not dissolve
- Pink is comparing with and without ascorbate, and also adding CNII (check)
- W/F media needs
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:
- HBSS (recipe below)
- dye solution in HBSS
- 4% glutaraldehyde (GAH) in HBSS
- Prepared that day from 50% GAH stock
- 1 sterile spatula per group
- 1 50ml conical tube for waste per hood
- 2 Petri dishes per group
- slides and coverslips
- 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