Figure 4
Experimental procedures for in vivo and ex vivo CETSA. Overview of CETSA sample preparation for both PBMC and tissues (spleen and brain) is described. (a) In vivo and ex vivo PBMC sample preparation. For in vivo evaluation, compounds are administered to C57BL/6 J mice. After the prescribed time, mouse peripheral blood is collected and PBMCs are isolated by density centrifugation over Ficoll according to the manufacturer’s instructions. For ex vivo evaluation, after the isolation of PBMCs with the Ficoll density centrifugation method, PBMCs are mixed with each compound. The isolated PBMC solutions are divided into 96-well PCR plates. The procedures for sample heat treatment and washing are described in Fig. 1. Both whole protein and supernatant samples are reserved for Western blot analysis. (b) In vivo spleen and brain sample preparation. Dissected spleen and brain samples are divided into four approximately equal parts. The samples are added to pre-warmed PBS containing protease inhibitor and incubated for 8 min at regulated temperatures. In the case of brain, the heat-treated samples are frozen using liquid nitrogen, and then homogenised with bead beaters. After bead homogenisation, the samples are freeze-thawed three times, and then homogenised by ultrasonic tissue homogenisation. For the spleen, preparation of the samples excluded initial sample freezing and ultrasonic tissue homogenisation. In the same way as PBMC sample preparation, both whole protein and supernatant samples are reserved for Western blot analysis. CETSA, cellular thermal shift assay; PBMC, peripheral blood mononuclear cell; PCR, polymerase chain reaction; WB, western blot.
