Cryopreserved PBMCs were thawed at 37⁰C in a water bath, washed with PBS (Lonza), filtered using 70μm cell strainers and counted. Next, PBMCs were stained with LIVE/DEAD Fixable Green Cell Stain Kit (Invitrogen) to exclude dead cells, following manufacturer's recommendations. Then, cells were washed with PBS containing 2.5% BSA (Sigma-Aldrich). Extracellular staining was carried out by incubating PBMCs for 30 min on ice in the dark with the following fluorochrome-conjugated monoclonal antibodies: PE anti-CD3 (UCHT1), PE anti-CD14 (MφP9), PE anti-CD19 (HIB19) and PE-Cy5 anti-CD56 (B159) from BD Bioscience and PEVio770 anti-NKp80 (4A4.D10) from Miltenyi Biotec. NK cells were identified as viable CD3-/CD14-/CD19-/NKp80+/CD56+/− cells (Orrantia et al., 2020 (link), 2021 (link)) and sorted using a BD FACSJazz cell sorter (Figure S4 ).
Pe cy5 anti cd56 b159
Manufactured by BD
Sourced in United States
PE-Cy5 anti-CD56 (B159) is a fluorescently labeled antibody that binds to the CD56 cell surface antigen. CD56 is expressed on natural killer cells, a subset of T cells, and some neural cells. This product can be used for the identification and enumeration of CD56-positive cells in flow cytometry applications.
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2 protocols using pe cy5 anti cd56 b159
1
Purification of Human NK Cells
2
Docetaxel-Induced NKG2D Expression
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MDAMB361 and BT474 cells were exposed to docetaxel (100 nM) for 6 to 72 hours or left untreated, collected and incubated at 4°C with anti-MICA, -MICB, -ULBP1, -ULBP2 (R&D Systems), followed by incubation with anti-mouse Alexa Fluor 448-conjugated reagent (Invitrogen, Waltham, MA, USA). Samples were analyzed by gating on live cells using the FACSCanto system (Becton-Dickinson, San Jose, CA, USA) and BD FACSDiva™ software (BD Bioscience).
PBMCs isolated from patients or healthy donors were analyzed using monoclonal antibodies to human antigens, including PE-anti-CD16 (3G8, BD Bioscience), PECy5-anti-CD56 (B159, BD Bioscience), APC-eFluor780-anti-CD3 (SK7, eBioscence, San Diego, CA, USA), FITC-anti-CD69 (FN50, BD Bioscience) and APC-NKG2D (ON72, Beckman Coulter, Miami, FL, USA).
Total splenocytes or purified Gr1+ myeloid cells from mouse spleens were analyzed using mouse monoclonal antibodies, including PE-anti-CD49b (DX5, eBioscience), APC-anti-NKG2D (CX5, eBioscience), PE-anti-CD11 (MI/70, BD Bioscience), FITC-anti-Ly6G (1A8, Mylteni Biotec) and APC-anti-Ly6C (HK1.4, eBioscience). Samples were analyzed by gating on physical parameters using the FACSCanto II system (Becton-Dickinson) and FlowJo software (Tree Star Inc, San Carlos, CA, USA).
PBMCs isolated from patients or healthy donors were analyzed using monoclonal antibodies to human antigens, including PE-anti-CD16 (3G8, BD Bioscience), PECy5-anti-CD56 (B159, BD Bioscience), APC-eFluor780-anti-CD3 (SK7, eBioscence, San Diego, CA, USA), FITC-anti-CD69 (FN50, BD Bioscience) and APC-NKG2D (ON72, Beckman Coulter, Miami, FL, USA).
Total splenocytes or purified Gr1+ myeloid cells from mouse spleens were analyzed using mouse monoclonal antibodies, including PE-anti-CD49b (DX5, eBioscience), APC-anti-NKG2D (CX5, eBioscience), PE-anti-CD11 (MI/70, BD Bioscience), FITC-anti-Ly6G (1A8, Mylteni Biotec) and APC-anti-Ly6C (HK1.4, eBioscience). Samples were analyzed by gating on physical parameters using the FACSCanto II system (Becton-Dickinson) and FlowJo software (Tree Star Inc, San Carlos, CA, USA).
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