20.109(S14): TA notes for module 3

General notes

revise for S14 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

Quiz.

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, vortexed, allowed to dissolve at 4 °C overnight in NaCl (150 mM), vortexed some more, then sterile filtered
   • Always put some NaCl in the conical first first, or dry alginate can get stuck/trapped and never dissolve
   • Don't try to make too much in one tube (>10-15 mL) of high wt %, or it may not uniformly dissolve

Day of Lab:

• Sterile-filter all alginate in the morning!
• Prepare base media with NEAA, HEPES, PSA; then add proline, ascorbate, and FCS/ITS as needed
• To be autoclaved, per group
  • 2 beakers (100 mL) for CaCl2 bath
  • 1 waste beaker (500 mL)
  • 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 → 40 mL conical per group
  • (4) 20ml aliquots of NaCl per group→ 40 mL conical per person
  • 2ml alginate w/excess
• 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

Media needs are now in Excel spreadsheet in Dropbox... but see example below for reference

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
   • use double recommended amount, i.e. 4 uL each dye per mL HBSS
3. 4% glutaraldehyde (GAH) in HBSS
   • Prepared that day from 50% GAH stock
4. 1 sterile spatula per group (ideally 2)
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
    • 10X TBS is 1M Tris, 2M NaCl, 50 mM CaCl2
  • Bring to pH 8.0 with 1 N NaOH, watching color rather than pH paper (pink, not yellow)
    • Add in 5 μL aliquots, typically 1-3 (most often 2) being needed
  • 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
• qPCR
  • water
  • qPCR plates
  • master mix prepared during class for each primer set
  • sterile reservoirs

Day of Lab:

• qPCR template: "ANS_07-2010"
• Quiz
• 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

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
  • D4902 Sigma
  • 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 or 20 μg/mL)
  • use at 1:500 in media (~102 nM)

• TGF PREP
  • 100 μg/mL in 10 mM citric acid, pH 3.0
  • citric acid monohydrate is 210.14 g/mol
  • also 0.1% BSA I think
  • Lily had done in 4 mM HCl/0.1% BSA

• FGF PREP
  • HH just did 100 μg/mL in PBS/0.1% BSA

• Papain Prep
  • Stock tends to be ~ 10U/mg and 25 mg/mL (always check lot)
  • Publication suggests 0.56 U/mL but I usually have doubled this
  • S14 for example: 5.2 mL buffer + 23.4 μL papain

• 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

• Linoleic acid just in case
  • ITS+1 has 1 mg/mL insulin, 0.55 mg/mL transferrin, 0.5 μg/mL NaSel, 50 mg/mL BSA, and 0.447 mg/mL LA
  • Ampule has 100 mg, and appears to be about 100 μL, so 1000 mg/mL
  • Suggests 2128x dilution, or 2.35 μL in 5 mL (enough for 200 mL, or 20 L media; but did sheet say enough for 5 L?)