Pe conjugated isotype control

U-CH2 and Chor-IN-1 cells were collected by trypsinization and recovered in their culture medium at 37 °C and 5% CO₂ for 30 minutes, then washed with PBS containing 1% FCS (staining buffer) and counted. 0.5 × 10⁶ cells of each cell line were resuspended in 100 µl staining buffer and stained with 10 µl PE-conjugated mouse anti-human CD24 or its correspondent PE-conjugated isotype control (BD Biosciences, San Jose, CA, USA) for 20 minutes at room temperature. Samples were washed with staining buffer, fixed with 1% formaldehyde for 10 minutes at 37 °C and permeabilized with 90% methanol for 30 minutes on ice. After washing with staining buffer, 10 µl of FITC-conjugated mouse anti-cytokeratin (CAM5.2) or its correspondent FITC-conjugated isotype control (BD Biosciences, San Jose, CA, USA) were added and incubated for 20 minutes at room temperature. Samples were washed with staining buffer, then acquired and analyzed with a FACSCalibur cytometer and CellQuest software (BD Biosciences, San Jose, CA, USA). Analysis was performed on 10,000 events, gating out debris and doublets.

MSCs isolated from cord blood were labeled with the following antibodies: FITC-conjugated human CD14 (cat# 555397), CD45 (cat# 555483), HLA-DR (cat# 555811), PE-conjugated human CD73 (cat# 550257), CD166 (cat# 559263, BD Biosciences), CD90 (cat# 12-0909-42), and CD105 (cat# 12-1057-42, Invitrogen). Isotype controls were also included: PE-conjugated Isotype Control (cat# 555743) and FITC-conjugated Isotype control (cat# 555573, BD Biosciences). Stained cells were analyzed by flow cytometry on a MACSQuant instrument (Miltenvi Biotec, Bergisch Gladbach, Germany).

Flow cytometry was used to assess the percentages of NGFR-positive cells in the melanoma cell population grown with and without trametinib. To exclude dead cells from analysis, cells were incubated with a LIVE/DEAD fixable Violet Dead Cell Stain Kit (Life Technologies, Eugene, OR, USA) for 30 min at 4 °C in the dark followed by three washes and staining with PE-conjugated antibodies (anti-NGFR #557196, BD Biosciences) for 45 min at 4 °C in the dark. An appropriate PE-conjugated isotype control (#555749, BD Biosciences) was included in each experiment. Three washes were performed prior to analysis using flow cytometer FACSVerse (BD Biosciences). Data were processed by BD FACSuite software.