Quantifying FATP1 and CD37 Colocalization

Antibody stainings for flow cytometry (MACS Quant, Miltenyi Biotec, Bergisch Gladbach, Germany) and confocal microscopy (LSM900, Zeiss, Jena, Germany) were performed on cells fixed with 2% paraformaldehyde and blocked with 5 µM FcBlock (Bd Biosciences, San Jose, CA, USA). Antibodies directed against FATP1 (308420, conjugated) (R&D Systems, Minneapolis, MN, USA, IC3304R), CD36 (877302, conjugated) (Novus Biologicals, Centennial, CO, USA, MAB19553AF647) and CD37 (HH1, unconjugated) (Santa Cruz Biotechnology, Dallas, TX, USA, sc-18881) were added as 10 µg/mL in PBS and stained for 30 min at RT in the dark. Isotype controls were added in similar concentrations. A secondary antibody against CD37 was used as 2,5 µg/mL in PBS and stained for 30 min at RT in the dark. For colocalization studies (LSM900, Zeiss, Jena, Germany), the Coloc2 function in ImageJ was used (Version 1.53 g)^{93 (link)}. Proximity ligation assays (PLA) were performed with FATP1 and CD37 antibodies according to the manufacturer’s instructions (Merck, Darmstadt, Germany). In addition, a second FATP1 antibody (polyclonal) (Novus Biologicals, Centennial, CO, USA, NBP2-69016) was used at 10 µg/mL as a positive control. CD37KO BJAB cells (generated by CRISPR/Cas9 knockout, described in^{92 (link)}) were used as a negative control in these experiments. Analysis was performed with ImageJ^{42 (link),94 (link)}.