Panserin 413 medium

Peripheral blood (PB) was collected from a healthy donor in a plastic Monovette EDTA tube. The isolation of human peripheral mononuclear cells (PBMCs) was executed using Histopaque^® (Sigma-Aldrich, St. Louis, MO, USA), as previously described [43 (link)]. Consequently, 3 mL of blood was carefully layered over 3 mL Histopaque^® and centrifuged at 400× g for 30 min at RT. The layer containing PBMCs at the interface between blood serum and Histopaque^® was transferred into a new tube and washed with PBS three times. The isolated cells were suspended in Panserin 413 medium (PAN-Biotech, Aidenbach, Germany) supplemented with 2.5% phytohemagglutinin M (PHA-M, Life Technologies, Darmstadt, Germany). Finally, the cell viability was measured using resazurin assay as described above.

Fresh peripheral blood samples were obtained from healthy donors and transferred into plastic Monovette EDTA tubes. The human peripheral mononuclear cells (PMNC) were isolated by Histopaque^® (Sigma-Aldrich, St. Louis, MO, USA), according to the previously described method [58 (link)]. Briefly, 3 mL blood was added carefully over 3 mL of Histopaque^® and centrifuged at 400× g for 30 min at 4 °C. Subsequently, the layer containing lymphocytes and PMNC was transmitted into a new tube and washed several times with PBS. Isolated cells were then maintained in a Panserin 413 medium (PAN-Biotech, Aidenbach, Germany) supplemented with 2% phytohemagglutinin M (PHA-M, Life Technologies, Darmstadt, Germany). Finally, the resazurin assay was performed to assess cell viability as described above.

The cell lines used in the present work, their origins and maintenance conditions were previously reported [60 (link)–63 (link)]. In brief, drug-sensitive CCRF-CEM and multidrug resistant P-glycoprotein-over-expressing CEM/ADR5000 leukemia cells, MDA-MB-231-pcDNA3 breast cancer cells and their transfectant subline MDA-MB-231-BCRP clone 23, HCT116 (p53^+/+) colon cancer cells and its knockout clone HCT116 (p53^-/-), U87.MG glioblastoma multiform cells and their transfectant subline U87.MGΔEGFR as well as HEK293 human embryonic kidney cells transfected with or without a cDNA for ABCB5 were used. The human peripheral mononuclear cells (PMNC) were isolated from fresh blood samples of a healthy donor by using Histopaque^® (Sigma-Aldrich, Taufkirchen, Germany).
Normal human peripheral mononuclear cells (PMNC) were isolated from fresh blood samples of a healthy donor using Histopaque^® (Sigma-Aldrich). In brief, 6 mL blood were layered with 6 mL Histopaque^® and centrifuged (400 × g) for 30 min at 4°C. The opaque interface containing lymphocytes and other mononuclear cells was transferred into a new tube and washed several times. Isolated PMNCs were kept in Panserin 413 medium (PAN-Biotech, Aidenbach, Germany) supplemented with 2 % phytohemagglutinin M (PHA-M, Life Technologies, Darmstadt, Germany).