Materials and Reagents

1. Tumour cells [in particular, this assay was set up for H1299 non-small cell lung carcinoma cells (ATCC, catalog number: CRL-5803 ) and breast cancer cell lines MDA-MB-231 (ATCC, catalog number: HBT-26 ),
   
2. MDA-MB-417.5 (kindly provided by Joan Massagué) and Hs578T (ATCC, catalog number: HBT-126 )]
   
3. Phosphate buffered saline (PBS) without Ca/Mg (any supplier)
   
4. Trypsin-EDTA (Life Technologies, Invitrogen^TM)
   
5. Fibronectin from bovine plasma (Sigma-Aldrich, catalog number: F1141 )
   
6. White carboxylate-modified latex beads (CML Latex Beads, 4% w/v, 0.4 µm), and number of particles is given in the MSDS/COA documents provided by the manufacturer (Thermo Fisher Scientific, catalog number: C37238 )
   
7. Black 96-well flat-bottom microscopy plates (µClear 96-well plate) (Greiner Bio-One GmbH, catalog number: 655090 )
   
8. 4% Formaldehyde (any supplier) in PBS
   
9. RPMI medium (Life Technologies)
   
10. Fetal Bovine Serum (FBS) (PAA Laboratories)
    
11. Penicillin-Streptomycin (Penicillin-Streptomycin) (Life Technologies, Gibco^®)
    
12. Complete medium (see Recipes)
    

Equipment

1. Cell culture set up, including laminar flow hood, cell culture incubator (37 °C and 5% CO₂), cell counter or counting chamber, multichannel pipets
   
2. BD Pathway 855 BioImager (BD Biosciences); or another brand or type of inverted microscope equipped with brightfield illumination, automated stage, 10x objective and software for automated imaging
   
3. Hydroflex plate washer (Tecan Trading AG)
   

Software

1. WIS-PhagoTracker v2_3_12 (Weizmann Institute of Science, http://www.weizmann.ac.il/vet/IC/software/wis-phagotracker; open source software)
   
2. Montage2MRC (Weizmann Institute of Science, http://www.weizmann.ac.il/vet/IC/software/wis-phagotracker; open source software)
   
3. KNIME (optional) (www.knime.org; open source software)
   

Procedure