Phycoerythrin pe

The study used bone marrow plasma cells from 109 patients with newly diagnosed MM and from 64 patients with MGUS. Plasma cells were purified from bone marrow mononuclear cells using anti-CD138 antibody conjugated with phycoerythrin (PE) (Beckman-Coulter, Brea, CA, USA) and an Easy Sep PE positive selection kit containing anti-PE antibody conjugated with micro-magnetic beads (STEMCELL Technologies, Vancouver, BC, Canada). The patients were diagnosed with MM or MGUS between July 2010 and March 2015. This study was approved by the institutional review board of Gunma University Hospital and followed all guidelines under the Declaration of Helsinki. Informed consent was obtained from all patients. The patients’ demographics are shown in Table 1.

Plasma cells were purified from bone marrow mononuclear cells with anti-CD138 antibody conjugated with phycoerythrin (PE) (Beckman Coulter, Brea, CA, USA) and the Easy Step PE positive selection kits containing anti-PE antibodies conjugated with micro-magnetic beads (STEMCELL Technologies, Vancouver, BC, Canada). RNA was extracted from the plasma cells (and one autopsied extramedullary plasmacytoma of the liver) and cell lines using mirVana RNA Isolation kits (Ambion, Austin, TX, USA). Complimentary DNA (cDNA) was produced using PrimeScript™ RT reagent kits with gDNA Eraser (TaKaRa Bio, Kyoto, Japan).

Rabbit polyclonal antibody anti-AE1 was kindly provided by Dr C. Wagner (University of Zürich, Switzerland). Mouse monoclonal antibody anti-AE1 (BRIC6) came from the International Blood Group Reference Laboratory (IBGRL, Bristol, United Kingdom). Mouse monoclonal antibodies anti-ankyrin R (clone N388A/10) and anti-AQP1 (clone 1/A5F6) were from Neuromab (UC Davis/NIH, USA) and Bio-Rad (Marnes-la-Coquette, France) respectively, while goat polyclonal anti-stomatin (M-14) and rabbit polyclonal anti-CAII antibodies were purchased from Santa Cruz Biotechnology (Dallas, Texas USA). Secondary antibodies used in flow cytometry analysis were phycoerythrin (PE) or fluorescein isothiocyanate (FITC)-conjugated F(ab’)₂ fragment of goat anti-mouse and donkey anti-goat immunoglobulins from Beckman Coulter (Villepinte, France). BCECF-AM [2′,7′bis-(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester] and pyranine [8-hydroxypyrene-1,3,6-trisulfonic acid] fluorescent probes, as well as carbonic anhydrase from bovine erythrocytes and the AE1 inhibitor DIDS [4,4 2-Diisothiocyanatostilbene-2,2 2-disulfonic acid disodium salt] were purchased from Sigma-Aldrich (Saint Quentin, France). Chloride indicator SPQ (6-methoxy-N-(3-sulfopropy1) Quinolinium) was obtained from Invitrogen (Fisher Scientific, Illkirch, France).