20.109(S13): TA notes for module 3

General notes

S13 notes: This experiment will be largely unchanged from the S12 version, with three potential exceptions: (1) Viability analysis will be done after two days, and final analysis after one week (instead of 7 and 12 days, respectively), pending IAP pilot experiments, perhaps with media being changed daily rather than every other day; (2) Students may be required (rather than encouraged) to replicate the work of another team and pool samples, with hopes of getting high enough concentrations for the protein and proteoglycan (PG) assays (3) Beads will be washed before the PG assay to limit interference from media (just use phenol red-free media?)

revise for S13 changes

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 in summer 2010:

• 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:

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 in NaCl (150 mM), 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 waste beaker
  • 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:

Ultimately just put on google document that year and in Spring 2012... but see example below

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 After Lab:

• For each ELISA sample
  • Add 1/10 of the starting volume of the digested solution (i.e., 20 μL) of 10X TBS pH 8.0
  • Bring to pH 8.0 with 1 N NaOH [already good enough usually?? do we add fixed amount?? checking with Han-Hwa]
  • Add another 1/10 V of elastase solution and incubate overnight in the fridge again

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)
• PG assay standards
  • samples thawed (from - 20)
  • 0.2 mg/mL chondroitin sulfate in 0.15% alginate, 250 μL per group
  • extra 0.15% alginate, about 0.9-1 mL per group
• PG assay dye
  • 0.021 g DMMB (di-methyl methylene blue) + 5 mL ethanol + 2 g sodium formate
  • after dissolve, take volume to 800 mL with distilled water
  • add formic acid to pH 1.5 (basically takes whole bottle in my experience. half the bottle should get you to 2.0)
  • volume to 1 L with distilled water

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)

• DEX PREP
  • MW 392.46
  • initial stock 1 mg/mL in ethanol (2.548 mM)
  • sub-dilute as 20 μL + 980 μL base medium and store frozen (50.96 μM)
  • use at 1:500 in media (~102 nM)

• TGF PREP

• 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

• FGF preparation
  • HH just did 100 mg/mL in PBS/0.1% BSA