For subcloning, neurospheres were collected and gently dissociated using papain (Worthington, Lakewood, NJ, USA) at 37 °C for 20 min with gentle agitation.
Lysates were clarified by centrifugation and incubated with His Select Beads (Sigma) for 30 min with gentle rocking at 4 °C.
Tumors were minced and digested with 2 mg/ml collagenase I (Sigma, C0130), 2 mg/mg hyaluronidase (Sigma, H3506) and 25 µg/ml Dnase (Promega) by incubating at 37 °C for 30 min with gentle mixing.
The centrifuge tube containing the mixture was incubated at 37°C for 15 min and 34°C for 105 min with gentle shaking to release protoplasts.
After 20 min with gentle agitation, the beads were transferred into 0.5% CaCl2 and process was repeated (20 min, gentle agitation).
After sterilization with 50% (w/v) commercial bleach for 30 min with gentle shaking, rice seeds were germinated on a Murashige Skoog medium under controlled conditions (28 °C/25 °C day/night, 8-h photoperiod, and 78% relative humidity).
Of the samples, 5 mg was re-dispersed in 10 mL of the organic dyes (concentration 10 mg/L) and the mixed solution was stored in the dark for 45 min with gentle stirring.
Other cell suspensions were incubated with Bio-TLS11a (100 nM) at 4 °C for 30 min, followed by SA-FSNPs (ca. 0.1 mg, 1 ml) at 37 °C for 60 min with gentle shaking.
The wells were washed thoroughly and then the secondary antibody conjugate solution was applied for 30 min with gentle agitation.
Proteins were eluted from beads by incubation in 1 M NH4OH at room temperature for 10 min with gentle agitation.
The binding of iodinated proteins was carried out at 4°C for 60 min with gentle rocking.
