Red blood lysis buffer

Peripheral blood was collected into ScreenCell blood collection tubes. 7.5 mL of each blood sample collected in a K2-EDTA was processed for 3 h using ScreenCell devices (Sarcelles, France) according to the manufacturer’s protocol. ScreenCell filters were washed with RPMI 1640 medium and Red Blood Lysis Buffer (MiltenyBiotec, Bologna, Italy). Then, enrichment filter-adherent CTCs were collected in RPMI medium and cell suspension filtered again. The ScreenCell microfiltration is an epitope-independent size-based device, able to capture all CTCs in the bloodstream. It has been used in CTC identification in rare tumors like hemangiopericytoma [22 (link)] as well as in more common tumors including bladder cancer [23 (link)].

After washing, hPCLS were cut into fine pieces in HBSS (Merck Sigma Aldrich) digestion buffer containing Collagenase Type IV 575 U/mL (Worthington), DNAse I 0.3 mg/mL (Merck Sigma Aldrich), Dispase II 2 U/mL (Merck Sigma Aldrich), Elastase 1.5 U/mL (Worthington) and CaCl2 5 mM (Merck Sigma Aldrich), and incubated for 1 h at 37 °C. The cells were filtered through a 40 µm cell strainer and the digestion was stopped with EDTA 50 mM and FBS 10%  v/v (Merck Sigma Aldrich) in DPBS. An erythrocyte lysis for 10 min at RT was performed by adding 1 mL Red Blood Lysis buffer (Miltenyi Biotec) to the cells.