Lymphocyte proliferative response to mitogens and the amount of TCR excision circles were determined as previously described (Amariglio et al., 2010 (link)). Proliferative response was measured by labeling PBMCs with 2.5 mM CFSE (Thermo Fisher Scientific), 7-aminoactinomycin D (BD Biosciences), PacB anti-CD3 (SK7; Biolegend), PE anti-CD25 (M-A251; BD Biosciences), APC-H7 anti-CD8 (SK1; BD Biosciences), and BV711 anti-CD4 (SK3; BD Biosciences) 4 d after stimulation. Activation response was determined by CD25 levels in stimulated PBMCs. The analysis of TCR Vβ expression was determined according to manufacturer’s manual (Beta Mark TCR Vβ Repertoire Kit; Beckman Coulter). NK degranulation was assessed by CD107a surface staining without stimulation (medium-cultured cells) and 3 h after stimulation with K562 cells at a ratio of 1:1 as previously described (Bryceson et al., 2012 (link)). The erythroleukemic cell line K562 (CCL-243; ATCC) was used as a target cell line. NK cells were cultured in medium containing 600 U/ml IL-2 (Novartis) for 48 h to assess the degranulation of activated NK cells. For surface staining, the following antibodies were used: PacB anti-CD3 (SK7; Biolegend), APC-H7 anti-CD8 (SK1; BD Biosciences), FITC anti-CD56 (NCAM16.2; BD Biosciences), and PE anti-CD107a (H4A3; BD Biosciences).
Pe anti cd107a h4a3
Manufactured by BD
Sourced in United States
PE anti-CD107a (H4A3) is a fluorescently labeled antibody that binds to the CD107a cell surface antigen. CD107a, also known as LAMP-1, is a lysosome-associated membrane protein that is expressed on the surface of cells during degranulation, such as in natural killer cells and cytotoxic T cells. The PE (phycoerythrin) fluorescent label allows for the detection and quantification of CD107a-expressing cells by flow cytometry.
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Lab products found in correlation
Beta mark tcr vβ repertoire kit, by Beckman Coulter (1 mentions)
Anti cd8 apc h7 sk1, by BD (1 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (1 mentions)
Interleukin 2 (il 2), by Novartis (1 mentions)
7 aminoactinomycin d, by BD (1 mentions)
Facsaria, by BD (1 mentions)
Anti cd3 apc h7 sk7, by BD (1 mentions)
Efluor 506, by Thermo Fisher Scientific (1 mentions)
Golgistop, by BD (1 mentions)
4 protocols using pe anti cd107a h4a3
1
Lymphocyte Proliferation and NK Cell Degranulation
2
NK Cell Degranulation and IFN-γ Assay
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The following mAbs were used: Percp-Cy5.5-anti-CD3 (UCHT1), APC-anti-CD56 (B159), PE-anti-CD57 (NK-1), PE-anti-NKp46(9E2/NKp46), PE-anti-CD158b (CH-L) and PE-anti-CD107a (H4A3) (from BD Biosciences); PE-anti-NKG2A (131411), AF700-, AF488- or purified anti-NKG2C (134591) (for the P815 coating used at 1 μg/mL, R&D Systems, Minneapolis, MN, USA); PE-Vio770-CD56 (AF12-7H3), PE-anti-CD16 (VEP13) (Miltenyi, Bergisch Gladbach, Germany); APC-anti-CD57 (HCD57; BioLegend, San Diego, CA, USA). NK cell degranulation assay was performed as previously described [12 (link)]. Cells were analyzed using FACSCalibur and sorted using FACSAria (BD Biosciences, San Jose, CA, USA). IFN-γ production was assessed in co-culture supernatants by ELISA kits (430104, Biolegend, San Diego, CA, USA).
3
Measuring NK Cell Degranulation
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NK cell degranulation was assessed as previously described [54 (link)]. Aliquots of one million freshly isolated PBMCs were incubated for 2 h at 37 °C in 200 µL complete RPMI-1640 medium under four stimulation conditions: leukocytes alone, +1 µg/mL elotuzumab, + MM.1R target cells (one million cells), or + both MM.1R and elotuzumab. Samples were centrifuged at 150 relative centrifugal force (RCF) for 3 min before incubation. Anti-CD107a-PE (H4A3, BD Biosciences Inc., San Jose, CA, USA), anti-CD45-PerCP-Cy5.5 (2D1, eBioscience, San Diego, CA, USA), anti-CD3-APC-H7 (SK7, BD Biosciences Inc., San Jose, CA, USA), and anti-CD56-APC (NCAM 16.2, Biolegend, San Diego, CA, USA) antibodies were added in the last 30 min of culture. Cells were centrifuged and rinsed twice with ice-cold wash buffer, with PI added in the second wash. NK cell degranulation was measured as a percentage of CD107a+ cells among total PI−CD45+CD3−CD56dim NK cells.
4
Evaluating Monalizumab's Impact on NK Cells
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To evaluate the e cacy of monalizumab to promote NK cell activity and ADCC, monalizumab and cetuximab were used. Monalizumab (purchased from Creative Biolabs, Shirley, NY, USA) is a humanized IgG4 mAb that targets NKG2A. Cetuximab is a chimeric IgG1 mAb targeting EGFR that induces ADCC.
Cell surface staining was performed using the following uorescently labeled mAbs: isotype control-PE (MOPC-2, RRID:AB_396091), anti-HLA-E-PE (3D12, RRID:AB_2869949) (eBioscience, San Diego, CA, USA), anti-CD107a-PE (H4A3, RRID:AB_396135), CD16-FITC (3G8 RRID: AB_395806, BD Biosciences, San Jose, CA, USA), anti-CD3-eFlour450 (17A2. AB_1272193, Invitrogen), anti-CD56-PE-Vio770 (REA196, RRID:AB_2726091), and anti-NKG2A-APC (REA110, RRID:AB_2726447, Miltenyi Biotec). To exclude dead cells, eFluor 506 (#65-0866-14, Invitrogen) staining was performed. Golgi-Stop (#554724, BD Biosciences) was used in the CD107a degranulation assay as a protein transport inhibitor.
Cell surface staining was performed using the following uorescently labeled mAbs: isotype control-PE (MOPC-2, RRID:AB_396091), anti-HLA-E-PE (3D12, RRID:AB_2869949) (eBioscience, San Diego, CA, USA), anti-CD107a-PE (H4A3, RRID:AB_396135), CD16-FITC (3G8 RRID: AB_395806, BD Biosciences, San Jose, CA, USA), anti-CD3-eFlour450 (17A2. AB_1272193, Invitrogen), anti-CD56-PE-Vio770 (REA196, RRID:AB_2726091), and anti-NKG2A-APC (REA110, RRID:AB_2726447, Miltenyi Biotec). To exclude dead cells, eFluor 506 (#65-0866-14, Invitrogen) staining was performed. Golgi-Stop (#554724, BD Biosciences) was used in the CD107a degranulation assay as a protein transport inhibitor.
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