20.109(S07): TA's notes for module 2

Ca Sensor Module

General notes

To get MES cell culture ready

For the first-day experiments

Sunday	Monday	Tuesday	Wednesday	Thursday	Friday	Saturday
	Thaw 106 MES cells (T25)	Exchange media	Exchange media	Passage MES cells (two T75s)	Exchange media	Exchange media
Split MES cells into 32 T25s (3x104 cells/cm2	Exchange media	Day 1 experiments	Day 1 experiments

Daily Notes

Notes:M2D1

1. There will be no quiz.
2. You will need at least 14 T-25 flasks of MES cells/lab as well as appropriate media for splitting cells. If more than 12 students are in lab section, add additional flask for each additional student.
3. Cells for microscope refresher can be from demo of trypsinization for first group on scopes and from students own trypsinized cells for second group on scopes.

Notes:M2D2

1. Need lab quiz.
2. Need 6 boxes of cuvettes
3. Need 12 96-well plates
4. Will need 6 flasks of MES cells/lab section as well as pre-transformation media for plating on slides.
5. Will need at least 12 4-well Lab-Tek slides per section.

Notes:M2D3

1. ?lab quiz
2. need to prep in advance some DNA to transfect

Notes:M2D5

fluo-3, AM
Here are experimental procedures:
To prepare 2 uM fluo-3 solution in Opti-MEM.

• Dissolve a vial of fluo-3 (50 ug) by adding 0.5 ml DMSO.
• Transfer all of the DMSO solution into 21.5 ml Opti-MEM in a Falcon tube.
• Mix it well and protect the solution from light.

To treat cells:

• Remove cell media from chambered slides where ES cells are growing.
• Add 1 ml of 2 uM fluo-3, AM
• Incubate at 37oC for 45 min (can do this at one time for all samples the class has prepared).
• Remove fluo-3, AM solution.
• Wash the cells with 1 ml warm PBS twice.
• Add 1 ml warm Opti-MEM.
• Incubate for 30 min or more.
• Students will observe and treat the cells under fluorescence microscope in the 109 lab.

Recipes/Reagents

1. Growth Media: 500 ml DMEM (high glucose), 50 ml FBS (Atlanta Biologic, Inc.), 5 ml P/S/G, 1 ml BME, 5 ml NEAA. Filter then add 50 ul LIF. Store 4°C
2. Pre-Tx’n Media: 500 ml DMEM (high glucose), 50 ml FBS (Atlanta Biologic, Inc.), 5 ml 100XG, 1 ml BME, 5 ml NEAA. Filter then add 50 ul LIF. Store 4°C
3. 0.1% gelatin for TC dishes > 10 min before adding cells: 1 g/L, Heat slightly to dissolve. Autoclave. Store 4°C
4. Cyproheptadine hydrochloride (C6022); Working conc should be 0.1 uM. Make stock of 0.1 M by dissolving 100 mg bottle in 3 ml DMSO (although according to Sigma this is water-soluble, I had trouble dissolving this in water). When using this stock dilute 1:1000 then final concentration of 1:1000 on cells. Store -20°C
5. A23187 Calcium Ionophore (C9275). Working concentration should be 2 uM. Make stock of 2 mM by dissolving 5 mg bottle in 4.6 ml DMSO. Use at 1:1000 on cells. Store -20°C
6. BAPTA-AM (A1076). Working concentration should be 30 uM. Make stock of 30 mM by dissolving 25 mg bottle in 1 ml DMSO. Use at 1:1000 on cells. Store -20°C
7. CaCl2*2H20 (Mallinckrodt 4160). Working conc should be 30 mM. Make stock of 3M by dissolving 4.41 g in 10 ml H20. Filter sterilize. Use at 1:100 on cells. Store -20°C
8. EGTA (JTBaker L657-05) Working concentration should be 0.5 mM. Make stock of 0.5 M by dissolving 1.9 g in 9 ml H20 + 1 ml 10 N NaOH. Filter sterilize. Use at 1:1000 on cells. Store -20°C
9. CaCl2 (no H20) for standard curve: 55 mg/50 ml H20 gives 10 mM solution.
10. Fluo-3, salt (F1240 from Molecular Probes). fw = 854.7g/mol. Dissolve 1 mg aliquot in 1.17 ml very clean H2O for 1 mM stock. Freeze 100 ul aliquots at -20°. Working concentration is 1 um (1:1000 dilution) for measuring free Ca2+ in vitro.
11. EGTA: fw 380.4 g/mol. 100 mM stock = 3.8 g/100 ml very clean H2O. pH with KOH to make soluble
12. KOH 10% solution (10 g/100 ml very clean H2O)
13. MOPS fw209g/mol 0.5M = 10.45g/100 ml very clean H2O, pH to 7.2 with HCl
14. KCl fw 74.5 g/mol 5 M stock (sat'd) = 37.25 g/100 ml very clean H2O, heat to dissolve or autoclave.
15. K2EGTA solution
    • 50 ml 100 mM K2EGTA
    • 10 ml 5M KCl
    • 30 ml MOPS
    • 410 ml v. clean H2O
16. CaEGTA
    • 0.367g CaCl2*2H2O dissolved in 250 ml K2EGTA solution