Anti mouse igg1 alexa fluor 647

List of anti‐human antibodies: PE‐CD133/1 (Clone AC133, Miltenyi Biotec), APC‐CXCR4 (Clone 12G5), FITC‐CD44 (Clone L178), PE‐CD166 (Clone 3A6) (all from BD Bioscience) and CD29‐APC (clone TS2/16, BioLegend) were incubated with cells for 30 minutes at 4°C.
Integrin β1 antibodies: anti‐mouse CD29 (Clone 9EG7‐550531 recognizing the active form of integrin β1), anti‐human CD49b (Clone 12F1‐555668), anti‐human CD49a (clone SR84 559 594) (all from BD Bioscience) were incubated with cells for 1 hour at room temperature (RT), then washed and incubated with secondary antibody (anti‐rat IgG2 Alexa Fluor 488 and/or anti‐mouse IgG1, Alexa Fluor 647, Thermo Fisher), for 30 minutes at RT.
Prior to FACS analysis, samples were incubated with 7‐AAD viability staining solution (10 μL/tube) (eBioscience) to exclude dead cells.
FACSCalibur and FACSCanto cell analyzers (Becton Dickinson) were used for data acquisition and FlowJo software V10 was used for data analysis.
LT73 CD133neg cell line was generated by sorting CD133 negative cells from the parental LT73 cell line using FACSAria cell sorter (BD Biosciences), as already reported.¹⁷Human disseminated tumor cells (DTC) were quantified in dissociated murine lung tissue, as 7AAD‐ live cells negative for anti‐mouse MHC class I (eBioscience), and their relative content of CD133+ CXCR4+ MIC was evaluated, as already detailed.¹⁷

Sixteen hours after transfection, HEK‐293 cells were fixed in 4% v/v paraformaldehyde (PFA) for 20 min and RAJI cells were adhered to poly‐l‐lysine‐coated coverslips and subsequently fixed. Cells were blocked in 3% w/v BSA, 1% w/v filtered human serum, and 10 mm glycine in PBS for 1 h and subsequently stained for 30 min in the same solution supplemented with 10 µg·mL⁻¹ of the following primary antibodies: anti‐CD9 (MEM61; Thermo Fisher Scientific) or anti‐EWI‐F (polyclonal; Thermo Fisher Scientific). This was followed by a 30‐min incubation with an appropriate secondary antibody: anti‐mouse IgG1‐Alexa Fluor 647 or anti‐rabbit IgG‐Alexa Fluor 647 (both Thermo Fisher Scientific). Samples were then stained with DAPI, washed, and embedded in mowiol. For intracellular staining of EWI‐F, cells were permeabilized for 10 min in 0.1% Triton X‐100 in PBS and all subsequent solutions contained 0.5% w/v saponin. Samples were imaged on a SP8 confocal microscope with a 60× water 1.2 NA objective (Leica, Wetzlar, Germany) using appropriate laser lines and settings. Colocalization was analyzed using the Coloc2 application of FIJI, and the other image analysis was performed using cellprofiler [92].

RAJI and HEK‐293 cells were harvested for flow cytometry analysis 16 h after transfection with GFP‐tagged CD9 (mutants). Single‐cell suspensions were blocked in PBS, 1% w/v BSA, and 2% v/v normal human serum for 30 min at 4 °C and subsequently stained for 30 min at 4 °C in the same solution supplemented with 10 µg·mL⁻¹ of the following primary antibodies: anti‐CD9 (MEM61; Thermo Fisher Scientific), anti‐CD9 (C9BB, a kind gift of M. E. Hemler), or mouse IgG1 (MOIC‐21; BioLegend, San Diego, CA, USA). This was followed by 30‐min incubation with anti‐mouse IgG1‐Alexa Fluor 647 (Thermo Fisher Scientific). Stained cells were analyzed using a Cyan flow cytometer (Beckman Coulter, Brea, CA, USA) and flowjo software version 10 (Tree Start Inc., Ashland, OR, USA).