Mononuclear cells were obtained from the bone marrow of newly diagnosed MM patients (n = 12), immediately after bone marrow aspiration. Next, myeloma cells were isolated using the CD138+ MACS system (Miltenyi Biotec Inc, Auburn, CA, USA). Isolated myeloma cells were then cryopreserved, enabling them to be used as an irradiated antigen-source for DCs, and as autologous primary myeloma cells. Isolated primary myeloma cells also were stained with mAbs against human CD38-PE, CD138-FITC, CD19-PE-Cy5.5, MHC class I-FITC, MICA-PE, MICB-PE, ULBP1-PE, ULBP-2/5/6-PE, HLA-E-PE, PD-L1-APC, CD112-PE, CD155-PE, and ICAM-1-APC (R&D System, USA) to determine morphology, the surface staining steps were described above. The samples were acquired using FACS Canto (USA), and the data were analyzed using FlowJo.
Ulbp1 pe
Manufactured by R&D Systems
Sourced in United States
ULBP1-PE is a recombinant Human ULBP1 protein that is labeled with Phycoerythrin (PE). ULBP1 is a member of the UL16-binding protein (ULBP) family and is involved in the activation of natural killer (NK) cells.
Automatically generated - may contain errors
Lab products found in correlation
Ulbp3, by R&D Systems (2 mentions)
Hla abc pe, by BD (2 mentions)
Cellquest software, by BD (2 mentions)
Alexa fluor 488 goat anti mouse igg, by Thermo Fisher Scientific (2 mentions)
Hla g fitc, by Bio-Rad (2 mentions)
Mica micb fitc, by Bio-Rad (2 mentions)
Cd155 fitc, by Bio-Rad (2 mentions)
Ulbp 2 pe, by R&D Systems (2 mentions)
Cd58 pe, by BD (2 mentions)
Tlr9 fitc, by Abcam (2 mentions)
Tlr4 pe, by Abcam (2 mentions)
Hla e, by Abcam (2 mentions)
Cd112, by Bio-Rad (2 mentions)
Ulbp 2 5 6 percp, by R&D Systems (2 mentions)
Cd54 pb hcd54, by BioLegend (2 mentions)
W6 32, by BioLegend (2 mentions)
Mica b pe cy7 6d4, by BioLegend (2 mentions)
B7h6 apc, by R&D Systems (2 mentions)
Facsaria 2, by BD (2 mentions)
Ulbp 2 5 6 pe, by R&D Systems (1 mentions)
6 protocols using ulbp1 pe
1
Isolation and Characterization of Primary Myeloma Cells
2
Evaluating Surface Marker Expression in MSC Variants
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The expression of surface markers on MSC, nvMSC-HLA-G1, and vMSC-HLA-G1 was evaluated by flow cytometry using the following monoclonal anti-human antibodies: HLA-G FITC (ABD Serotec, Oxford, UK), HLA DR, DP, DQ FITC (BD Bioscience, San Jose, CA), HLA-ABC PE (BD Pharmingen, San Jose, CA), MICA/MICB FITC, CD155 FITC (both from ABD Serotec), ULBP-1 PE, ULBP-2 PE (both from R&D Systems), CD58 PE (BD Biosciences), TLR9 FITC, TLR4 PE (both from Abcam, Cambridge, UK) and TLR3 Alexa 647 (Imagenex, Port Coquitlam, BC, Canada). Cells were also stained for HLA E (Abcam), ULBP-3 (R&D Systems), and CD112 (ABD Serotec) using Alexa Fluor 488 Goat Anti-Mouse IgG (Molecular Probes-Life Technologies) as secondary antibody. Briefly, cells were incubated for 15 minutes in the dark with saturating concentrations of each antibody. Stained cells were then washed with phosphate-buffered saline 0.1% azide, fixed with 1% formaldehyde, and analyzed by flow cytometry using the CellQuest software (Becton Dickinson Biosciences, San Jose, CA). A total of 10,000 events was acquired for each sample. Appropriate isotype (negative) controls were performed for each antibody.
3
Exosome Isolation and Identification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Exosomes were isolated from serum using Izon Science qEV Size Exclusion Columns according to the manufacturer's instructions. For identification of exosomes in serum, Fc receptor blocking reagent (Miltenyi) was added to paired serum, exosome and protein fractions before staining with BODIPY-FL (ThermoFisher) and ULBP1-PE (R&D). Samples were fixed with Cytofix (BD) before acquisition. Samples were acquired by ImagestreamXL (Amnis) and data analysis performed using IDEAS software (Amnis).
4
Selinexor Modulates NK Cell Ligands
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Ramos, SU-DHL-4 and JeKo-1 cells were incubated with selinexor (50-2000nM), leptomycin B (50nM) or DMSO control for 16 hours in the presence of QVD. Cells were then surface stained with antibodies against activating NK cell ligands Vimentin-A488 (clone 280618, R&D Systems), ULBP-1-PE (170818, R&D Systems), ULBP-2/5/6-PerCP (165903, R&D Systems), CD54-PB (HCD54, Biolegend), B7H6-APC (875001, R&D Systems) and MICA/B-PE/Cy7 (6D4, Biolegend); the inhibitory NK cell ligand HLA-E-PE/Cy7 (3D12, Biolegend) and pan-HLA class-I molecules (W6/32, Biolegend) for 30 min at 4°C. CLL cells were incubated with selinexor (50-2000nM) or DMSO control for 40 hours in the presence of QVD then incubated with 10% human serum at 4°C for 15 minutes before CD5+CD19+ CLL cells were surface stained with anti-HLA-E-PE/Cy7 (3D12, Biolegend) for 30 min at 4°C. Normal PBMC were incubated with selinexor (500-2000nM) or DMSO control for 16 hours then surface stained with anti-HLA-E-APC (3D12, Biolegend), anti-CD3-PerCP (UCHT1, Biolegend), anti-CD19-PE (HIB19, Biolegend) and anti-CD56-PE/Cy7 (HCD56, Biolegend) for 30 min at 4°C. Cells were then acquired on a BD FACS Aria II (BD Biosciences) using FACSDiva software (BD Biosciences) as above.
5
Multiparametric Flow Cytometry Profiling of CLL Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After treatment with selinexor, leptomycin B or brefeldin A (BFA, GolgiPlug, BD Bioscience) for the required timepoint, Fc receptors were blocked on CLL cells with 10% human serum for 15 min then cells were stained with CD19-PE (HIB19, Biolegend) or CD19-PB, CD5-PerCP (UCHT2, Biolegend) or CD5-APC, ULBP-1-PE (170818, R&D Systems), ULBP-2/5/6-PerCP (165903, R&D Systems), CD54-PB (HCD54, Biolegend), B7H6-APC (875001, R&D Systems), ecto-calreticulin-A488 (EPR3924, Abcam), MICA/B-PE/Cy7 (6D4, Biolegend), CD20-PE (2H7, BioLegend), CD38-A488 (HIT2, BioLegend), FAS-FITC (DX2, Biolegend), DR4-PE (DJR1, Biolegend), DR5-APC (DJR2-4, Biolegend), HLA-E-PE/Cy7 (3D12, Biolegend) and total HLA class I proteins (W6/32, Biolegend) for 30 min at 4 °C. Assessment of CLL cells that have recently egressed from the lymph nodes was performed as previously described [30 (link), 31 (link)]. Patient CLL cells were rested for 1 h at 37 °C, then stained with CD19-BV510 (HIB19), CD5-APC, HLA-E-PE/Cy7 (3D12), pan-HLA (W6/32) and CXCR4-PE (12G5, Biolegend). Cells were then acquired on a BD FACS Aria II.
6
Evaluating Surface Marker Expression in MSC Variants
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!