Novocyte novosampler pro

Sixty high affinity AXL binding Pronectins were identified by screening 1 µM Pronectin on MDA-MB-231 (Axl expression) and 293 cells (No Axl expression) using a Novocyte NovoSampler Pro flow cytometer (Acea Biosciences). The Pronectins were ranked by ratio MDA MFI vs. 293 MFI. In a U-Bottom Polystyrene plate, 0.1 × 10⁶ cells/well were seeded in 100 µL of FACS buffer (1× PBS + 5% FBS) for each cell line. Pronectin was added to each well at 1 µM and plates were incubated at 4 °C for 30 min. Plates were spun down at 1500 rpm and washed two times with FACS Buffer. Then, 1 µL of Anti-HIS Alexa Flour 4888 (R&D Systems) and 1 µL of Anti-hAXL APC-conjugated Antibody (R&D Systems) were added for 30 min at 4 °C. The samples were spun down at 1500 rpm for 3 min and washed two times with FACS Buffer. Finally, 100 µL of FACS buffer was added and the cells were analyzed by flow cytometer.

Cells were dissociated into a single-cell suspension using Versene for undifferentiated iPSCs or Accutase for differentiated cells and fixed using the Foxp3/Transcription Factor Staining Buffer Set (eBioscience, San Diego, CA, USA), according to the manufacturer’s instructions. Cellular marker quantification was performed using NovoCyte NovoSampler Pro (Acea Biosciences, Santa Clara, CA, USA), utilizing FlowJo^TM software (BD Life Sciences, Franklin Lakes, NJ, USA). Primary antibodies used were anti OCT3/4 (sc-2004, 1:100, Santa Cruz, Dallas, TX, USA), SOX1 (ab87775, 1:500, Abcam, Cambridge, UK), NESTIN (ABD69, 1:500, Sigma-Aldrich, Saint Louis, MO, USA), TUJ1α (T8660, 1:1000, Sigma-Aldrich, Saint Louis, MO, USA), GFAP (ab7260, 1:1000, Abcam) ISL1 (ab178400, 1:100, Abcam, Cambridge, UK), MAP2ab (M1406,1:500, Sigma-Aldrich, Saint Louis, MO, USA) or ChAT (GTX113164, 1:500, GeneTex, Irvine, CA, USA). Secondary antibodies were goat anti-mouse Alexa Fluor 488 (A-11029, 1:500, Thermo Fisher, Waltham, MA, USA), goat anti-Rabbit Alexa Fluor 633 (A-21071, 1:500, Thermo Fisher, Waltham, MA, USA).