Cell Culture Conditions

CELL CULTURE CONDITIONS

MDA MB435 -

Growth Media - MEM/EBSS (Minimal Essential Media with Earles Basal Salts Solution Gibco/BRL) supplemented with 5% FBS (HyClone), 2mM Glutamine (Sigma), 1X (vol/vol) Non Essential Amino Acids (Sigma) , 2X (vol/vol) MEM Vitamins (Sigma), 1mM Sodium Pyruvate (Sigma), 100 IU/ml penicillin/100µg/ml streptomycin (Gibco/BRL).

Experimental Media - same as above with 2% serum instead of 5%.

Defined Media - MEM/EBSS supplemented with 1.25 mg/ml pyrogen poor BSA (Pierce), 10 µg/ml transferrin, 10 µg/ml insulin, 2mM Glutamine, 100 IU/ml penicillin/100µg/ml streptomycin (optional).

Subculture of Cells: Remove media from flask of cells (they should be 70-80% confluent). Place approximately 5 ml of pre-warmed, 1XTrypsin/1mM EDTA in flask and rock the flask so that the trypsin covers the entire area of cells, then remove and discard trypsin (do not trypsinize for more than 30 seconds). Let flask stand for 2-3 minutes then rinse cells from flask with pre-warmed media. Do a 1:10 split on the cells if they were 70-80% confluent (e.g. rinse cells off with 10 ml of media then remove 9 ml of cell suspension). Add media to a final volume of 20 ml in the T75 flasks. Return to incubator. Note: these cells require overnight to re-adhere to the flask.

MDA MB231 -

Growth Media - MEM/EBSS (Minimal Essential Media with Earles Basal Salts Solution Gibco/BRL) supplemented with 10% FBS (HyClone), 2mM Glutamine (Sigma), 1X (vol/vol) Non Essential Amino Acids (Sigma) , 1mM HEPES, 100 IU/ml penicillin/100µg/ml streptomycin (Gibco/BRL).

Experimental Media - same as above with 5% serum instead of 10%.

Subculture of Cells: Remove media from flask of cells (they should be 70-80% confluent). Place approximately 5 ml of pre-warmed, 1XTrypsin/1mM EDTA in flask and rock the flask so that the trypsin covers the entire area of cells, then remove and discard trypsin (do not trypsinize for more than 30 seconds). Let flask stand for 2-3 minutes then rinse cells from flask with pre-warmed media. Do a 1:5 split on the cells if they were 70-80% confluent. Add media to a final volume of 20 ml in the T75 flasks. Return to incubator.

MCF-7 McGuire -

Experimental Media - same as above with 5% serum instead of 10%.

Subculture of Cells: Remove media from flask of cells (they should be 70-80% confluent). Place approximately 5 ml of pre-warmed, 1XTrypsin/1mM EDTA in flask and rock the flask so that the trypsin covers the entire area of cells, incubate for 1 minute, then remove and discard. Let flask stand for 5 minutes then rinse cells from flask with pre-warmed media. Do a 1:5 split on the cells if they were 70-80% confluent. Add media to a final volume of 20 ml in the T75 flasks. Return to incubator.

T47D -

Growth Media - DMEM High Glucose (DulbeccoÕs Modified Eagles Media) supplemented with 10% FBS (HyClone), 2mM Glutamine (Sigma), 100 IU/ml penicillin/100µg/ml streptomycin (Gibco/BRL).

Experimental Media - same as above with 5% serum instead of 10%.

Subculture of Cells: Remove media from flask of cells (they should be 70-80% confluent). Place approximately 5 ml of pre-warmed, 1XTrypsin/1mM EDTA in flask and rock the flask so that the trypsin covers the entire area of cells, incubate for 1 minute, then remove and discard. Let flask stand for 5 minutes then rinse cells from flask with pre-warmed media. Do a 1:5 split on the cells if they were 70-80% confluent. Add media to a final volume of 20 ml in the T75 flasks. Return to incubator.

MCF-10A -

Growth Media - DMEM/F12 (DulbeccoÕs Modified Eagles Media/F12 HamÕs ) supplemented with 5% Horse Serum (HyClone), 2mM Glutamine (Sigma), 1X (vol/vol) antibiotic/antimycotic (pen/Strep/ ampB), 100 ng/ml cholera toxin, 0.5 ng/ml hydrocortisone, 10µg/ml insulin, 20 ng/ml EGF.

Experimental Media - same as above with 2% serum instead of 5%.

Subculture of Cells: Remove media from flask of cells (they should be 70-80% confluent). Rinse with BSS (Basal Salt solution) and remove rinse. Add 9 ml of BSS to flask and 1 ml of 10X trypsin. Incubate for 5 minutes, remove and discard. Add another 9 ml of BSS and 1ml of 10X trypsin to the flask and incubate for 10 minutes. Remove (keep and centrifuge if cells have come off in the trypsin solution). Rinse cells off of flask with pre-warmed media and do a 1:10 split on the cells. Add media to a final volume of 20 ml in a T75 flask.

BSS - per liter

8 g NaCl

0.2 g K phosphated (monobasic)

0.2 g KCl

1 g dextrose

1.15 g Na phosphate (dibasic)

Sterile filter and store at 4¡C.