5.2 2013

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)

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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

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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

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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

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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