Ficoll paque solution

Peripheral blood samples were obtained from patients at the time of AKI. All samples were collected at Mayo Clinic’s central laboratory and PBMCs were processed in the research laboratory. PBMCs were isolated using density-gradient centrifugation (Ficoll-Paque solution) (GE Healthcare Biosciences) and cells were then slowly frozen to −80 °C and subsequently to less than −196 °C in liquid nitrogen for batch analysis.

Fresh lung cancer tissues and Blood samples for peripheral blood mononuclear cells (PBMC) extraction of patients were obtained with the donor’s written consent and approved by Zhejiang Cancer Hospital Committee (IRB-2020–324). PBMC were obtained by density gradient centrifugation using Ficoll-Paque solution (GE Healthcare, 17–1,440–02, Uppsala, Sweden). H292 cells were inoculated into 96 well plates (3,000–5,000 cells/well) and then treated with Bafetinib for 24 h. PBMC cells were activated with anti-CD3/CD28/CD2 (50 µL antibody was added to every 2 ml dish) for 3–5 days in advance, and then co-incubated according to the ratio of PBMC: tumor cells 5:1. After 8–12 h, drain the culture solution with a pipette, dye it with crystal violet at room temperature for 30 min, wash it with PBS until there is no floating color, then add 100 µL PBS and take photos with a microscope.

Samples of human primary MM cells were obtained under Institutional Review Board approved protocols at our institution (University of Fukui). Residual bone marrow aspiration samples was obtained from patients with previously untreated MM. Mononuclear cells were separated using ficoll-paque solution (GE Healthcare). Then, the mononuclear cells were mixed with Cellbanker (Takara Bio Company, Japan) and stored in deep freezer. Each sample contained more than 80% of MM cells.

For study purposes, samples were collected from six woman aged 25 ~ 35 years old with the informed consent approved by the Ethics Committee of Shengjing Hospital affiliated to China Medical University. MenSCs were then isolated and cultured accordingly as our previous study reported [30 (link)]. In brief, MenSCs were purified from menstrual blood by density gradient centrifugation with Ficoll-Paque solution (GE Healthcare Bio-sciences, Uppsala, Sweden). The enriched MenSCs were cultured in 25-cm² culture flask with DMEM/F12 medium (Hyclone, Logan, UT, USA) supplemented with 10% FBS (Gibco, Waltham, MA, USA), 2 mM L-glutamine (Sigma-Aldrich), 1% non-essential amino acid (Sigma-Aldrich) and 1% penicillin-streptomycin (Sigma-Aldrich) in a humidified incubator under 37 °C with 5% CO₂.

In this experimental study, CD133⁺ HSCs were isolated from three samples of umbilical cord blood. A mononuclear cell fraction from cord blood was isolated by Ficoll-Paque solution (GE Healthcare Bio-sciences AB, Sweden) and centrifuged in 400g for 30 minutes at 22˚C. To remove the platelets, the cell pellet was centrifuged at 200 g for 10 minutes at 22˚C. Then, the pellet was resuspended in 500 μL of phosphate buffered saline (PBS, Medicago AB, Sweden). 50 μL of FcR blocking reagent (Miltenyi Biotec GmbH, Germany) was added, mixed well and incubated at 2-8˚C for 10 minutes. Afterwards, 50 μL of CD133 microbeads (Miltenyi Biotec GmbH, Germany) were added to the cells and incubated for 30 minutes at 4˚C. The Cells were centrifuged at 300 g for 5 minutes. The supernatant was aspirated and the cells were re-suspended in 500 μL of PBS. The cell suspension was added to a positive selection column. Column was washed with PBS. The column was removed from the magnetic separator and placed on a suitable collection tube. Enough amount of buffer was pipetted onto the column. After that, the magnetically labeled cells were flush outed by tightly pushing the plunger into the column.

Peripheral blood samples were obtained from patients pre-transplantation, at 3, 6 and 12 months post-transplant. Samples were all collected between 7–9am and were processed within 4 hours at the Immunological Core Facility at the Transplant Research Center, Brigham and Women's Hospital. PBMCs were isolated by density gradient centrifugation using Ficoll-Paque solution (GE Healthcare Biosciences) spun at 800 g for 30 minutes at 20°C.