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 &mu;L volume (of papain/digestion buffer) for a good signal
 * Implement qPCR instead of semi-quantitative gel method
 * in progress

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 &deg;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 &mu;L 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 &mu;L, 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 &mu;L per 6 mL
 * 1 group adding double ascorbate, means 3 &mu;L 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 &mu;L per 12 mL, or 6 &mu;L 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
 * 4) *Prepared that day from 50% GAH stock
 * 5) 1 sterile spatula per group
 * 6) 1 50ml conical tube for waste per hood
 * 7) 2 Petri dishes per group
 * 8) slides and coverslips
 * 9) 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 &mu;L 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 + &beta;-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 &mu;L
 * (1) 300ul aliquot of CN II standard per group
 * prep 3.5 mL plus 35 &mu;L
 * 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 &mu;L 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 &mu;M)
 * Stock: 11.5 mg/mL in DMEM, freeze single-use aliquots
 * HEPES (10 mM)
 * Ascorbate(20 &mu;g/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 &mu;M)
 * HEPES (10 mM)
 * Chondrogenic factors
 * TGF-beta1 (10 ng/mL)
 * Dexamethasone (100 nM)
 * Ascorbate (40 &mu;g/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