20.109(S07): TA's notes for module 2: Difference between revisions
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= | =Cacium Sensor Module= | ||
==General notes== | ==General notes== | ||
In order to prepare MES cells for this module, you need to prepare 10 vials each having 10<sup>6</sup> 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. | In order to prepare MES cells for this module, you need to prepare 10 vials each having 10<sup>6</sup> 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. | ||
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<b><sup>1)</sup></b> You must prepare frozen vials of MES cells ahead of time. | <b><sup>1)</sup></b> You must prepare frozen vials of MES cells ahead of time. | ||
<b><sup>2)</sup></b> 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. | <b><sup>2)</sup></b> 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. | ||
<b><sup>3)</sup></b> Recommended cell seeding densities: 3x10<sup>4</sup> cells/cm<sup>2</sup> for the T/Th group and 10<sup>4</sup> cells/cm<sup>2</sup> for the W/F group | <b><sup>3)</sup></b> Recommended cell seeding densities: 3x10<sup>4</sup> cells/cm<sup>2</sup> for the T/Th group and 10<sup>4</sup> cells/cm<sup>2</sup> for the W/F group. Note that plating efficiency of MES cells onto gelatinized polystyrene surface is usually less than 50%. | ||
===Notes:M2D2=== | ===Notes:M2D2=== | ||
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## 6 flasks of MES cells per lab section | ## 6 flasks of MES cells per lab section | ||
Table. Cell preparation schedule for day 2 experiments | |||
{| border=1px | |||
|'''Sunday''' | |||
|'''Monday''' | |||
|'''Tuesday''' | |||
|'''Wednesday''' | |||
|'''Thursday''' | |||
|'''Friday''' | |||
|'''Saturday''' | |||
|- | |||
|Thaw two vials of MES cells <sup>1)</sup> | |||
|Exchange media | |||
|Split MES cells into 32 4-well Lab-Tek slides | |||
|Exchange media | |||
|Day 2 experiments | |||
|Day 2 experiments | |||
| | |||
|} | |||
<b><sup>1)</sup></b> Cell seeding density = 2 x 10 <sup>6</sup> cells. Use a gelatinized T-75 flask. | |||
===Notes:M2D3=== | ===Notes:M2D3=== | ||
# | # You need to prepare lab quiz. | ||
#need to | # You need to prepare in advance some DNA to transfect. | ||
===Notes:M2D4=== | ===Notes:M2D4=== | ||
# No lab quiz | |||
# Pre-experiments with Fluo-3, AM in the morning | |||
# 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. | 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. | * Dissolve a vial of fluo-3 (50 ug) by adding 0.5 ml DMSO. | ||
* Mix it well and protect the solution from light. | * 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: | To treat cells: | ||
* Remove cell media from chambered slides where ES cells are growing. | |||
* Add 1 ml of 2 uM fluo-3, AM | * Remove cell media from chambered slides where ES cells are growing. | ||
* Incubate at 37oC for 45 | * 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=== | |||
#You need to prepare lab quiz. | |||
===Notes:M2D6=== | ===Notes:M2D6=== | ||
==Recipes/Reagents== | ==Recipes/Reagents== | ||
# 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 | # 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 | ||
# Pre- | # 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 | ||
# 0.1% gelatin for TC dishes > 10 min before adding cells: 1 g/L, Heat slightly to dissolve. Autoclave. Store 4°C | # 0.1% gelatin for TC dishes > 10 min before adding cells: 1 g/L, Heat slightly to dissolve. Autoclave. Store 4°C | ||
# 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 | # 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 | ||
# 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 | # 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 | ||
# 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 | # 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 | ||
# | # CaCl<sub>2</sub>*2H<sub>2</sub>0 (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 | ||
# 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 | # 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 | ||
# | # CaCl<sub>2</sub> (no H<sub>2</sub>0) for standard curve: 55 mg/50 ml H<sub>2</sub>0 gives 10 mM solution. | ||
# Fluo-3, salt (F1240 from Molecular Probes). | # Fluo-3, salt (F1240 from Molecular Probes). Fw = 854.7g/mol. Dissolve 1 mg aliquot in 1.17 ml very clean H<sub>2</sub>O for 1 mM stock. Freeze 100 ul aliquots at -20°. Working concentration is 1 um (1:1000 dilution) for measuring free Ca<sup>2+</sup> <i>in vitro</i>. | ||
# EGTA: | # EGTA: Fw 380.4 g/mol. 100 mM stock = 3.8 g/100 ml very clean H<sub>2</sub>O. pH with KOH to make soluble | ||
# KOH 10% solution (10 g/100 ml very clean | # KOH 10% solution (10 g/100 ml very clean H<sub>2</sub>O) | ||
# MOPS | # MOPS Fw 209 g/mol 0.5M = 10.45g/100 ml very clean H<sub>2</sub>O, pH to 7.2 with HCl | ||
# KCl fw 74.5 g/mol 5 M stock (sat'd) = 37.25 g/100 ml very clean | # KCl fw 74.5 g/mol 5 M stock (sat'd) = 37.25 g/100 ml very clean H<sub>2</sub>O, heat to dissolve or autoclave. | ||
# <b>K2EGTA solution </b> | # <b>K2EGTA solution </b> | ||
#*50 ml 100 mM K2EGTA | #*50 ml 100 mM K2EGTA | ||
#*10 ml 5M KCl | #*10 ml 5M KCl | ||
#*30 ml MOPS | #*30 ml MOPS | ||
#*410 ml v. clean | #*410 ml v. clean H<sub>2</sub>O | ||
#<b>CaEGTA</b> | #<b>CaEGTA</b> | ||
#*0.367g | #*0.367g CaCl<sub>2</sub>*2H<sub>2</sub>O dissolved in 250 ml K2EGTA solution |
Latest revision as of 20:02, 18 March 2007
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
- There will be no quiz.
- 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.
- 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.
- 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
- You need to prepare lab quiz.
- You need to prepare the following materials
- 6 boxes of polystyrene cuvettes
- 12 96-well tissue culture plates
- At least 12 4-well Lab-Tek slides per lab section.
- Pre-transformation media for plating on slides.
- 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
- You need to prepare lab quiz.
- You need to prepare in advance some DNA to transfect.
Notes:M2D4
- No lab quiz
- Pre-experiments with Fluo-3, AM in the morning
- 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
- You need to prepare lab quiz.
Notes:M2D6
Recipes/Reagents
- 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
- 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
- 0.1% gelatin for TC dishes > 10 min before adding cells: 1 g/L, Heat slightly to dissolve. Autoclave. Store 4°C
- 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
- 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
- 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
- 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
- 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
- CaCl2 (no H20) for standard curve: 55 mg/50 ml H20 gives 10 mM solution.
- 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.
- EGTA: Fw 380.4 g/mol. 100 mM stock = 3.8 g/100 ml very clean H2O. pH with KOH to make soluble
- KOH 10% solution (10 g/100 ml very clean H2O)
- MOPS Fw 209 g/mol 0.5M = 10.45g/100 ml very clean H2O, pH to 7.2 with HCl
- KCl fw 74.5 g/mol 5 M stock (sat'd) = 37.25 g/100 ml very clean H2O, heat to dissolve or autoclave.
- K2EGTA solution
- 50 ml 100 mM K2EGTA
- 10 ml 5M KCl
- 30 ml MOPS
- 410 ml v. clean H2O
- CaEGTA
- 0.367g CaCl2*2H2O dissolved in 250 ml K2EGTA solution