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

Cacium Sensor Module

General notes

In order to prepare MES cells for this module, you need to prepare 10 vials each having 10⁶ frozen cells ahead of time. You will thaw the vials whenever you need to provide cells to students. The time table below may help you set up your schedule.

Daily Notes

Notes:M2D1

1. There will be no quiz.
2. 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.
3. 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.
4. If your target cell density is ca. 10⁵ cells/cm², you need to spread at least 3X10⁴ cells/cm² a couple of days before experiments.

Table. Cell preparation schedule for day 1 experiments

Sunday	Monday	Tuesday	Wednesday	Thursday	Friday	Saturday
	Thaw 106 MES cells (T25) 1)	Exchange media	Exchange media	Passage MES cells (two T75s)	Exchange media	Exchange media
Split MES cells into 32 T25s 2), 3)	Exchange media	Day 1 experiments	Day 1 experiments

¹⁾ You must prepare frozen vials of MES cells ahead of time. ²⁾ Fourteen flasks are enough for each lab section, but it might be good to prepare 2 ~ 3 extra flaks because students can make a mistake and can ask you extra ones. Note that you will also use two flasks for demonstration. ³⁾ Recommended cell seeding densities: 3x10⁴ cells/cm² for the T/Th group and 10⁴ cells/cm² for the W/F group. Note that plating efficiency of MES cells onto gelatinized polystyrene surface is usually less than 50%.

Notes:M2D2

1. You need to prepare lab quiz.
2. You need to prepare the following materials
   1. 6 boxes of polystyrene cuvettes
   2. 12 96-well tissue culture plates
   3. At least 12 4-well Lab-Tek slides per lab section.
   4. Pre-transformation media for plating on slides.
   5. 6 flasks of MES cells per lab section

Table. Cell preparation schedule for day 2 experiments

Sunday	Monday	Tuesday	Wednesday	Thursday	Friday	Saturday
Thaw two vials of MES cells 1)	Exchange media	Split MES cells into 32 4-well Lab-Tek slides	Exchange media	Day 2 experiments	Day 2 experiments

¹⁾ Cell seeding density = 2 x 10 ⁶ cells. Use a gelatinized T-75 flask.

Notes:M2D3

1. You need to prepare lab quiz.
2. You need to prepare in advance some DNA to transfect.

Notes:M2D4

1. No lab quiz
2. Pre-experiments with Fluo-3, AM in the morning
3. You need to prepare many different solutions for testing calcium sensors

Experiments you need to do before the lab.

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 at least 45 mi (You can do this just once for all samples around noon).
   * Students will remove fluo-3, AM solution.

Notes:M2D5

1. You need to prepare lab quiz.

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-Transformation 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 Fw 209 g/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