Pierce concentrators 150 k mwco

His-tagged TOP1 was expressed in pET-28(a) vector in BL21. His-Tagged TOP2 was expressed in pET-32 vector in BL21. After the induction of His-tagged protein production, a crude extract of proteins were subjected to HisPur Cobalt Column purification (Thermo Scientific). The proteins were further purified using size-exclusion chromatography. Additional purification and concentrations were done using the Amicon Ultra 50 k Centrifuge Filter (EMD Millipore) in the case of the monomer. Pierce Concentrators 150 k MWCO (Thermo Scientific) was used to further purify and concentration the dimer. Small aliquots were stored or utilized in the same day.

B16F0 melanoma cells were used as a source of tumor antigens, and LLC cells were used as a control to determine the level of nonspecific stimulation (Figure 2). The cells were collected from the plates, centrifuged at 200× g 5 min, resuspended in PBS at a concentration of 2–3 × 10⁶ cells/mL and lysed by 5–6 cycles of rapid freezing in liquid nitrogen (−196 °C) and heating in a water bath (37 °C). Cell debris was removed from the resulting suspension by centrifugation.
In a series of experiments to increase the immunogenicity of lysate tumor, cells were exposed to heat shock before lysis. The plates with cells were placed in a thermostat at 41 °C for 1 h. Then, the culture medium and the tumor cells were collected, cells were lysed, and the culture medium was centrifuged at 1000× g 10 min to remove the cell debris. The supernatant was concentrated in 20 mL concentration tubes with a permeable membrane for proteins with a molecular weight of less than 150 kDa (Pierce Concentrators 150K MWCO, Thermo Scientific, Waltham, MA, USA) to harvest heat-shock-induced microvesicles. The protein concentrations in the lysate and in the microvesicle preparation were determined using the colorimetric method based on bicinchoninic acid (Pierce BCA Protein assay kit, Thermo Scientific).