BM was prepared by flushing two femurs with 500 μl PBS. After pelleting the cells, supernatants were used for BM extra-fluid measurements and stored at −80 °C. For detecting and quantifying proteins, commercially available ELISA kits for IL-1α (R&D Systems, DY400), IL-1β (R&D Systems, DY401) and IL-6 (R&D Systems, DY406) were used.
Supernatants from transduced CD34+ HPCs were collected at different time points (days 7, 9 and 14) and stored at −80 °C. For measurement of proteins levels, commercially available ELISA kits for IL-1α (BioLegend, 445807), IL-1β (BioLegend, 437007), IL-6 (BioLegend, 430507) and IL-8 (BioLegend, 431507) were used.
For human plasma specimens, blood was collected from pre-therapy pediatric patients with multisystem LCH and healthy pediatric controls in EDTA tubes and centrifuged at 400g for 30 min over a Ficoll gradient, and then the plasma supernatant was collected and recentrifuged for 5 min, and then purified supernatant was stored at −80 °C. None of the plasma samples analyzed underwent more than two freeze–thaw cycles. Protein levels were determined using the MAGPIX instrument (Luminex). The concentration of each analyte was measured by comparison to the protein standards. Levels of IL-6 and IL-8 were measured with the Millipore Human Cytokine/Chemokine Panel I (1:2 dilution).
Supernatants from transduced CD34+ HPCs were collected at different time points (days 7, 9 and 14) and stored at −80 °C. For measurement of proteins levels, commercially available ELISA kits for IL-1α (BioLegend, 445807), IL-1β (BioLegend, 437007), IL-6 (BioLegend, 430507) and IL-8 (BioLegend, 431507) were used.
For human plasma specimens, blood was collected from pre-therapy pediatric patients with multisystem LCH and healthy pediatric controls in EDTA tubes and centrifuged at 400g for 30 min over a Ficoll gradient, and then the plasma supernatant was collected and recentrifuged for 5 min, and then purified supernatant was stored at −80 °C. None of the plasma samples analyzed underwent more than two freeze–thaw cycles. Protein levels were determined using the MAGPIX instrument (Luminex). The concentration of each analyte was measured by comparison to the protein standards. Levels of IL-6 and IL-8 were measured with the Millipore Human Cytokine/Chemokine Panel I (1:2 dilution).