VivosX for MCF10A Cells

Cells

Examples of cell lines (Tet-inducible) used:

MCF10A / pBABE-hygro-rtTA / pLT-H2AZ-H43C-V5-iGSP 

MCF10A / pBABE-hygro-rtTA / pLT-H2AZ-KKRR-H43C-V5-iGSP

Reagents

MCF10A Media

• Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12)
• 5% horse serum
• 20 ng/ml human recombinant epidermal growth factor (EGF)
• 0.5 µg/ml hydrocortisone
• 0.1 µg/ml cholera toxin
• 10 µg/ml insulin
• 50 U/ml penicillin and 50 U/ml streptomycin

4- DPS stock

Prepare 360 mM in DMSO (1:2,000)

• Aldrithiol-4 (MW: 220.31 g/ml, Sigma cat #143057)
• Dissolve 40 mg (0.040 g) in 500 µl of DMSO
• Store at -20C

http://www.sigmaaldrich.com/catalog/product/aldrich/143057?lang=en&region=US

NEM stock

Prepare 2 M stock in DMSO (1:40)

• NEM (MW: 125.13, Sigma #E1271)
• Dissolve 0.25 g in 1 ml DMSO
• Store at -20C

http://www.sigmaaldrich.com/catalog/product/sial/e1271?lang=en&region=US

TUNES Lysis Buffer

Stir for 30 min or until all components have dissolved. Filter sterilize and store at room temperature.

Add fresh before use:

Procedure

For a 6-well plate. Three wells for 4-DPS crosslinking and the other three wells for DMSO control. (Triplicate samples)

1. Seed 100,000 cells in 2 ml growth medium per well of a 6-well plate.
2. Allow cells to attach overnight. Induce with 1 µg/ml doxycycline the next morning. Induce cells for 48 hr.
3. Treat cells with 180 µM 4-DPS or DMSO at 37C for 20 min. 4-DPS stock: 360 mM in DMSO (1:2,000)
   • Thaw 4-DPS at RT. Vortex and spin before use.
   • Prepare two 15 ml conical tubes, each with 7 ml of 10A growth media (pre-warm to 37C).
   • Add 3.5 µl of 360 mM 4-DPS or 3.5 µl of DMSO.
   • Mix.
   • Aspirate media from cells and add 2 ml of 4-DPS/DMSO medium.
   • Incubate at 37C for 20 min.
   • During the incubation, label seven 2-ml microtubes for harvest and keep them on ice.
4. Wash cells twice with cold PBS on ice.
   • Aspirate media.
   • Rinse with 2 ml cold PBS.
   • On last wash, tap the plate lightly to ensure all excess PBS is removed.
   • Put the plate on ice immediately after wash.
5. Harvest cells.
   • Prepare lysis buffer in a pre-chilled 2-ml microtube. 
   • Thaw NEM at RT. (Stock: 2 M in DMSO, 1:40)
   • Need 200 µl lysis buffer per well. (Adjust vol. based on the cell confluency) 
   • Add NEM and Halt phosphatase inhibitor fresh to the lysis buffer.
   • 1351 µl TUNES lysis buffer + 35 µl NEM + 14 µl Halt inhibitor cocktail
   • Add 200 µl of lysis buffer per well.
   • Keep the plate on ice while adding the lysis buffer.
   • Incubate on ice for 15 min.
   • Scrap the cells and collect into pre-chilled 2-ml microtubes.
6. Snap freeze cell lysates and store at -80C or proceed to protein harvesting.