Prostatosphere Formation Assay Protocol

Prostatosphere formation assays were performed using a slight modification of previously described techniques [39 (link), 40 (link)]. Cells were plated in DMEM F-12 (11320-033, Life Technologies) containing 10 ng/mL bFGF (233-FB/CF, R&D Systems, Minneapolis, MN), 20 ng/mL EGF (236-EG, R&D Systems), 5 mg/mL insulin (3435, R&D Systems), and 0.4% (v/v) bovine serum albumin (BSA) (5217, R&D Systems) supplemented with 1% (v/v) knockout serum replacement (10828-028, Life Technologies) at 500-3,000 cells per well in 6-well ultra low attachment plates. In some case, the cultures were treated with the Mer inhibitor UNC1062 (AOB4488, AOBIOUS, Gloucester, MA). Prostatosphere formation (cell clusters of 10 cells or greater) was observed at 7–10 days under a light microscopy.

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Shiozawa Y., Berry J.E., Eber M.R., Jung Y., Yumoto K., Cackowski F.C., Yoon H.J., Parsana P., Mehra R., Wang J., McGee S., Lee E., Nagrath S., Pienta K.J, & Taichman R.S. (2016). The marrow niche controls the cancer stem cell phenotype of disseminated prostate cancer. Oncotarget, 7(27), 41217-41232.

Publication 2016

DMEM F-12 EGF (236-EG, UNC1062

Assays Bovine serum albumin Cells Insulin Light microscopy Serum Unc1062

Corresponding Organization :

Other organizations : Wake Forest University, University of Michigan–Ann Arbor, Johns Hopkins University

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Variable analysis

independent variables

Treatment with the Mer inhibitor UNC1062

dependent variables

Prostatosphere formation (cell clusters of 10 cells or greater)

control variables

DMEM F-12 medium
10 ng/mL bFGF
20 ng/mL EGF
5 mg/mL insulin
0.4% (v/v) bovine serum albumin (BSA)
1% (v/v) knockout serum replacement
Plating density (500-3,000 cells per well)
Observation time (7–10 days)

controls

Positive control: Prostatosphere formation in the absence of the Mer inhibitor UNC1062
Negative control: Not specified

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