5.2 2013

Submitted by kdorfman on Thu, 09/26/2013 - 14:46

M 10/28

Bulk-phase endocytosis

** 2 coverslips, 2 small diameter MatTek dishes 3t3

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

  • 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 http://wahoo.nsm.umass.edu/content/paraformaldehyde-fix

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