bmsc human cell line hs 5  (ATCC)

Journal: American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons

Article Title: Proteasomal adaptations underlying carfilzomib-resistance in human bone marrow plasma cells

doi: 10.1111/ajt.15634

Figure Lengend Snippet: Effect of in vitro carfilzomib treatment on β5i levels in BMPCs. Patient BMPCs (100,000 cells) were co-cultured with BMSC conditioned media for 72 h in the presence or absence of carfilzomib for 72 h. Cells were removed, pelleted and lysates prepared in 50 uL celLytic non-denaturing buffer. Histone 3 was used as a loading control. Lysates were separated on 4-12% bis-tris gels, transferred to PVDF membranes and probed using Abs to proteasome subunit β5i (PSMB8) (A) and β5 (PSMB5) (B). Erythrocytes (RBCs) contain constitutive 20S proteasomes and do not contain i20S proteasomes. RBC lysates was used as positive control to detect constitutive 20S proteasomes. Ratio indicates the relative intensity of a given proteasome subunit in lysates of that individual patient prior to and after carfilzomib treatment (N=8).

Article Snippet: CD138 + BMPCs were co-cultured with conditioned media from the human HS.5 BMSC line (ATCC, Manassas, VA) supplemented with APRIL and IL-6 for indicated times.

Techniques: In Vitro, Cell Culture, Control, Positive Control

Journal: American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons

Article Title: Proteasomal adaptations underlying carfilzomib-resistance in human bone marrow plasma cells

doi: 10.1111/ajt.15634

Figure Lengend Snippet: CD138+ BMPCs from HLA-sensitized patients produce class I and II Abs at levels comparable to those detected in patient serum. Class I and II Ab levels were detected, identified and titered in serum from HLA-sensitized patients and culture supernatants from BM cell fractions using the single antigen bead (SAB) assay (LABScreen®; One Lambda, Canoga Park, CA) assay on a Luminex platform (Austin, TX). BM mononuclear cells (20,000/assay), CD138+ (100,000/assay) and CD138− PCs (100,000/assay) from that same patient were incubated in 100 uL BMSC conditioned media supplemented with APRIL and IL-6 at 37°C for 72 h. Serum and cell culture supernatants were removed and 20 uL was used for SAB assays (48, 49). A representative histogram of the Luminex™ SAB results to determine the levels of class I and II HLA in patient serum and cell fractions is shown. Similar results were observed using serum and cells from two additional HLA-sensitized patients (see Supp. Figure 5A, B).

Article Snippet: CD138 + BMPCs were co-cultured with conditioned media from the human HS.5 BMSC line (ATCC, Manassas, VA) supplemented with APRIL and IL-6 for indicated times.

Techniques: Luminex, Incubation, Cell Culture

Journal: American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons

Article Title: Proteasomal adaptations underlying carfilzomib-resistance in human bone marrow plasma cells

doi: 10.1111/ajt.15634

Figure Lengend Snippet: Effect of carfilzomib treatment in vitro on the production of class I (A.) and II (B.) Abs by BMPCs from HLA-sensitized patients. Patient BMPCs (100,000 cells) were co-cultured in 100uL BMSC conditioned media for 72 h. Cell suspensions were centrifuged for 3 min at 3,000 rpm, culture supernatants removed and individual class I and II Ab levels measured using the SAB assay. Culture media was incubated alone as a negative control. Serum isolated from the whole blood of HLA-sensitized patients was used to measure the levels of individual class I and II Abs.

Article Snippet: CD138 + BMPCs were co-cultured with conditioned media from the human HS.5 BMSC line (ATCC, Manassas, VA) supplemented with APRIL and IL-6 for indicated times.

Techniques: In Vitro, Cell Culture, Incubation, Negative Control, Isolation