5.3 2013

5.3 2013 kdorfman Thu, 09/26/2013 - 14:47

W 10/30

Transport of endosomes along microtubules

3 small-diameter MatTek dishes of LLCPk GFP tubulin

HBS-BSA 1mg/mL

  • to mix transferrin ( 3 mL) and nocodazole (15 mL) in

  • to rinse growth medium off before and after treatment. 6 mL per dish x 3 dishes per group x 8 groups = 144 mL. Aliquot 20 mL.

  • make 250 mL

To make mL HBS-BSA
add: mL 10X HBS
add: g BSA
plus water to final volume

Fe-HBS-BSA
to mix transferrin in
Need 2.3 mL; make 3 mL

Solution stock conc final conc 1 2 2.5 3 4 mL
Fe-citrate (mM) 89.4 0.1 1.118 2.237 2.796 3.35 4.47 µL

in Fe-HBS-BSA to final volume

MAKE ENOUGH OF EACH FOR EACH GROUP TO DO 3 TREATMENTS

Transferrin in Fe-HBS-BSA
Alexa-Fluor 488 transferrin (Invitrogen T13342, MW = ~80KD)
stock is 5 mg/mL (=62.5 µM).
Final concentration = 1 µM.

  • Each group may do up to 3 100 µL treatments (= 3 x 7 x 105 µL = 2.205 mL)
  • Make 105 µL aliquots (21)
  • Make 2.3 mL: 36.8 µL AF-488-Tf + 2.263 mL Fe-HBS-BSA

if new transferrin stock is needed add 1 mL sterile distilled water to the new bottle.

# aliquots 1 2 3 6 9 12 15 18 21
vi 62.5µM tf 1.68 3.36 5.04 10.1 15.1 20.2 25.2 30.2 35.3 µL
HBS-Fe-BSA 103.3 206.6 310 620 930 1240 1550 1860 2170 µL
Vf 1 µM tf 105 210 315 630 945 1260 1575 1890 2205 µL

Nocodazole
Acros 358240100, 10 mg
1µM in HBS-BSA
(Working concentration range is usually 100nM - 30 µM.)
Stock is 10 mg/mL (=33mM)
(Add 1mL DMSO to 10 mg in bottle = 33mM. Aliquot 3.03 µL. To make 1mM, add 97 µL.)

Make enough for each group to do two 1 mL treatments.
Aliquot 1.01 mL per tube, 15 tubes = 15 mL. Make 16 mL

  • Add 97 µL HBS-BSA to 3 µL 33mM in tube to make 1 mM
  • Mix 1 part 1mM with 999 parts HBS-BSA to make 1µM: 16 mL + 1.6 µL
# aliquots 1 2 3 4 5 10 15
vi 1 mM noc 1.01 2.02 3.03 4.04 5.05 10.1 15.15 µL
HBS-BSA 1.009 2.018 3.027 4.036 5.045 10.09 15.135 mL
vf 1 µM noc 1.01 2.02 3.03 4.04 5.05 10.1 15.15 mL

non-CO2 medium
to mix TMR-dextran in
to incubate cells in

TMR-dextran

in non-CO2 medium
Each group may do up to 3 treatments = 3 x 7 x 100 µL = 2.205 mL.

Invitrogen D3308, 10 mg
Make 100 mg/mL stock (add 100µL to the bottle)
1 µL aliquots

TMR-dextran Working concentration = 0.05 mg/mL in medium
100 µL per treatment
Aliquot 105 µL, make enough for each group to do 3

Make 10 mg/mL dilution to make student aliquots
1 µL 100 mg/mL stock + 9µL medium

no. aliquots 1 2 3 6 9 12 15 18 21
vi (10 mg/mL TMR-dex) 0.525 1.05 1.575 2.2 3.15 4.2 6.3 7.875 11.025 µL
medium 104.5 128.9 656.7 875.6 1094.5 1313.4 1532.3 1751.2 µL
vf (0.05 mg/mL TMR-dex) 105 210 315 630 945 1260 1575 1890 2205 µL