Reagents for 6.3

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 7 groups = 126mL. Aliquot 20 mL.

• make 200 mL

ingredient	10	20	25	30	50	75	100	200	250	mL
10 x HBS	1	2	2.5	3	5	7.5	10	20	25	mL
BSA	0.01	0.02	0.025	0.03	0.05	0.075	0.1	0.2	0.25	g

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

---

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