6.2: Bulk endocytosis
6.2: Bulk endocytosis kdorfman Mon, 10/17/2011 - 17:05Bulk-phase Endocytosis
Cells for 6.2 2012
Cells for 6.2 2012 kdorfman Fri, 10/19/2012 - 12:323t3
2 coverslips per group
2 small diameter MatTeks per group
Cells for 6.2
Cells for 6.2 kdorfman Mon, 10/17/2011 - 17:09Lab is Monday 10/24/11
cells on coverslips: Students should use the same cell type as for Lab 6.1
- 3t3 8 (2 per group for 4 groups)
- LLCPk "
cells on MatTek dishes
(small diameter, so 100 µL will be enough to treat the cells):
- 3t3 8 (2 per group for 4 groups)
- LLCPk "
Reagents for 6.2
Reagents for 6.2 kdorfman Mon, 10/17/2011 - 20:29Monday 10/24
Students use the same cell type as for Lab 6.1
PBS for rinsing live cells WARM
PBS for rinsing fixed cells RT
Take from the supply of sterile aliquots
Growth media for incubation WARM
10 mL aliquots each
- F10-Hams for LLCPk cells
- DMEM for 3t3 cells
- non-CO2 medium for observing live cells (could use this for all cells for simplicity's sake)
TMR-dextran to stain endosomes in cells on coverslips
Invitrogen D3308, 10 mg
Make 10 mg/mL stock (add 1 mL to the bottle)
Make 8.8 µL aliquots in 2 mL tubes.
TMR-dextran Working concentration = 0.05 mg/mL in medium
2 coverslips at 50 µL/coverslip (=~110 µL/group)
Add 1751.2 µL medium to 8.8 µL 10mg/mL stock to make 0.05 mg/mL working solution.
Aliquot ~105 µL/group
#aliquots | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | |
---|---|---|---|---|---|---|---|---|---|
vi (10 mg/mL TMR-dex) | 1.1 | 2.2 | 3.3 | 4.4 | 5.5 | 6.6 | 7.7 | 8.8 | µL |
medium | 218.9 | 437.8 | 656.7 | 875.6 | 1094.5 | 1313.4 | 1532.3 | 1751.2 | µL |
vf | 220 | 440 | 660 | 880 | 1100 | 1320 | 1540 | 1760 | µL |
TMR-dextran/ FITC-dextran to stain endosomes in cells in MatTeks
tmr as above
FITC-dextran Working concentration = 0.5 mg/mL in medium
(1mg/mL might be better, but that uses up a whole bottle.)
2 dishes at 100 µL/dish (=~220 µL/group)
Add 1742.4 µL medium to an 8.8 µL aliquot of 10 mg/mL TMR-dex plus 8.8 µL 100 mg/mL FITC-Dex
#aliquots | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | |
---|---|---|---|---|---|---|---|---|---|
vi (10 mg/mL TMR-dex) | 1.1 | 2.2 | 3.3 | 4.4 | 5.5 | 6.6 | 7.7 | 8.8 | µL |
vi (100 mg/mL FITC-dex) | 1.1 | 2.2 | 3.3 | 4.4 | 5.5 | 6.6 | 7.7 | 8.8 | µL |
medium | 217.8 | 435.6 | 650.1 | 871.2 | 1089 | 1306.8 | 1524.6 | 1742.4 | µL |
vf | 220 | 440 | 660 | 880 | 1100 | 1320 | 1540 | 1760 | µL |
Methylamine solution (1M)
Sigma 426466-100mL 40% w/vol (=12.9M; MW=31) ~$25
(Working concentration ~20mM - ~20µL/mL. Students need ~100 µL)
Make 1 mL, aliquot ~120 µL
Solution | stock conc | final conc | 1 | 5 | 10 | 15 | 20 | mL |
---|---|---|---|---|---|---|---|---|
10X PBS | 10 | 1 | 0.1 | 0.5 | 1 | 1.5 | 2 | mL |
methylamine | 12.9 | 1 | 78 | 388 | 775 | 1163 | 1550 | µL |
plus water to final volume
Fixative
- PBS-paraformaldehyde 3.7%
- Need 70 mL
- Divided in 2 aliquots, one per fume hood
Ingredient | 10 | 15 | 20 | 25 | 40 | 50 | 60 | 70 | mL final |
---|---|---|---|---|---|---|---|---|---|
paraformaldehyde | 0.37 | 0.555 | 2 | 0.925 | 1.48 | 1.85 | 2.22 | 2.59 | g |
water | 9 | 13.5 | 18 | 22.5 | 36 | 45 | 57.78 | 63 | mL |
10X PBS | 1 | 1.5 | 2 | 2.5 | 4 | 5 | 6 | 7 | mL |
heat paraformaldehyde in water, then add 10X HBS
Add a drop of 10N NaOH
Heat and stir in the fume hood
OR
Ingredient | 10 | 15 | 20 | 25 | 40 | 50 | 60 | 70 | mL final |
---|---|---|---|---|---|---|---|---|---|
32% paraformaldehyde | 1.16 | 1.73 | 2.31 | 2.89 | 4.63 | 5.78 | 6.94 | 8.09 | mL |
10X PBS | 1 | 1.5 | 2 | 2.5 | 4 | 5 | 6 | 7 | mL |
water | 7.84 | 11.77 | 15.69 | 19.61 | 31.38 | 39.22 | 47.06 | 54.91 | mL |