# Phage-Phenotype Imaging of Myeloma Plasma Cells by Phage Display
## 2 Materials and Methods
### Phase Selection and Binding Affinity  
A 9-mer M13-pVIII random phage display library was constructed in the vector pC89, by cloning a random DNA insert in-frame of encoding segments of the pVIII gene [31]. The procedure described in [PERSON] et al. [32] was used to isolate specific phage clone against multiple myeloma cell line (MM.1). The MM.1 cell line was used because several cell phenotypes expressing mixed surface markers are present. First, the phage library was used in a pre-selection against plastic materials, used for the selection protocol against MM.1 cells. Non-binding phage were used in four rounds of affinity selection against MM.1 cells (1 \(\times\) 10\({}^{6}\) cells/mL in [PERSON]'s buffered salt solution, Sigma-Aldrich; pH 7.6). Each round included the incubation of phage with the target for 1 h at room temperature (RT) with gentle agitation. Cells-phage complex was precipitated by spinning (8000\(\times\) g, 5 min) and separated from unbound phage by five-times washing and centrifugation steps (8000\(\times\) g, 5 min) with 1 mL 0.1%TBS/Tween (50 mM Tris-HCl, pH 7.5; 150 mM NaCl; 0.1% Tween-20). Phage were finally eluted from the cells-phage complex by 250 \(\mathrm{\SIUnitSymbolMicro L}\) of 0.2 M glycine-HCl (pH 2.2) in gentle shaking at RT for 10 min. Then, the solutions were neutralized with 25 \(\mathrm{\SIUnitSymbolMicro L}\) of 1 M Tris-HCl (pH 9.1).  
Phage clones from the last round of affinity selection were identified by plating infected _E. coli_ TG1 cells on LB agar added with X-Gal (5-bromo-4-chloro-3-indolyl-beta-d-galactopyranoside), IPTG (isopropyl thiogalactoside), and ampicillin. The presence of the insert was detected by X-gal, which produces a characteristic blue dye when cleaved by \(\beta\)-galactosidase. Blue bacterial colonies, from the fourth round of selection, were randomly selected, considering that each colony contained a single phage clone. The binding ability of selected phage clones to the target was tested in ELISA using as a negative control vector pC89. Briefly, 2.5 \(\times\) 10\({}^{4}\) MM.1 cells in phosphate-buffered saline (PBS) were dispensed in each well of a 96-well plate and incubated overnight at 4 \({}^{\circ}\)C. After the incubation, cells were fixed by methanol (10 min at room temperature), washed three times with washing buffer (PBS + 0.05% Tween 20), and blocked with 100 \(\mathrm{\SIUnitSymbolMicro L}\) of PBS + 5% lactalbumin + 0.05% Tween 20 (2 h at 37 \({}^{\circ}\)C in slow shaking 30 rpm). The 1 \(\times\) 10\({}^{10}\)/100 \(\mathrm{\SIUnitSymbolMicro L}\) phage clones, in PBS + 1% lactalbumin + 0.1% Tween 20, were added to each well, and the plate was incubated for 1 h at 37 \({}^{\circ}\)C in slow shaking 30 rpm. Then, cells were washed three times with washing buffer and incubated with monoclonal anti-M13 peroxidase conjugate antibody (Amersham Biosciences, Buckinghamshire, UK) at a dilution of 1/5000 in PBS + 1% lactalbumin + 0.1% Tween-20 (100 \(\mathrm{\SIUnitSymbolMicro L}\)/well) for 1 h at 37 \({}^{\circ}\)C. Finally, cells were washed three times with washing buffer and incubated for 45 min at room temperature with 100 \(\mathrm{\SIUnitSymbolMicro L}\) of 3,3\({}^{\prime}\),5,5\({}^{\prime}\)-Tetra-methylbenzidine (TMB) for antibody binding detection. Reaction was stopped with 25 \(\mathrm{\SIUnitSymbolMicro L}\) of 1 M H\({}_{2}\)SO\({}_{4}\) and optical absorbance recorded at 450 nm using a MultiSkan FC ThermoScientific.