Anti cd107a phycoerythrin pe cy5

Manufactured by BD

Anti-CD107a-phycoerythrin (PE)-Cy5 is a fluorescently-labeled antibody that binds to the CD107a cell surface marker. CD107a is a lysosomal-associated membrane protein (LAMP-1) that is expressed on the surface of cells during degranulation, a process associated with the release of lytic granules. The PE-Cy5 fluorescent label allows for the detection and analysis of CD107a-expressing cells using flow cytometry.

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Lab products found in correlation

Brefeldin a, by Merck Group (8 mentions) Golgistop, by BD (8 mentions) Anti cd56 pe cy7, by BD (8 mentions) Anti ifn γ apc, by BD (7 mentions) Anti cd3 alexafluor700, by BD (7 mentions) Anti mip1β pe, by BD (7 mentions) Anti cd16 allophycocyanin apc cy7, by BD (7 mentions) Rosettesep, by STEMCELL (7 mentions) Fix and perm a and b solutions, by Thermo Fisher Scientific (3 mentions) Nunc maxisorp flat bottom, by Thermo Fisher Scientific (2 mentions) Fix and perm a and b, by Thermo Fisher Scientific (2 mentions) Anti cd16 apc cy5, by BD (1 mentions) Anti cd3 pacblue, by BD (1 mentions)

Close spelling variants of this product

Anti-CD107a PE-Cy5 CD107a-PE/Cy5 Anti-CD107a-PE CD107a-PE Phycoerythrin (PE) Anti-CD107a

8 protocols using anti cd107a phycoerythrin pe cy5

Assay for NK Cell Activation Markers

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An assay to determine NK cell activation (NKA) state based on the expression of surface CD107a and intracellular production of IFNγ and MIP1β was performed as previously described^{3 (link)}. NK cells were isolated from whole blood from healthy donors using negative selection with RosetteSep (STEMCELL Technologies), added to a SIV_mac239 gp120-absorbed plate with purified IgG, anti-CD107a-phycoerythrin (PE)-Cy5 (BD), brefeldin A (Sigma), and GolgiStop (BD), and incubated for 5 hrs at 37°C. After the incubation, cells were stained for surface markers using anti-CD16-allophycocyanin (APC)-Cy7 (BD), anti-CD56-PE-Cy7 (BD), and anti-CD3-Alexa Fluor 700 (BD) and then stained intracellularly with anti-IFNγ-APC (BD) and anti-MIP1β-PE (BD) after treatment with Perm A and B solutions (Invitrogen). Cells were then fixed in 4% paraformaldehyde and analyzed by flow cytometry. NK cells were defined as CD3-negative and CD16-positive and/or CD56-positive, and the percent of NK cells positive for each marker was determined.

Ackerman M.E., Das J., Pittala S., Broge T., Linde C., Suscovich T.J., Brown E.P., Bradley T., Natarajan H., Lin S., Sassic J.K., O’Keefe S., Mehta N., Goodman D., Sips M., Weiner J.A., Tomaras G.D., Haynes B.F., Lauffenburger D.A., Bailey-Kellogg C., Roederer M, & Alter G. (2018). Route of immunization defines multiple mechanisms of vaccine-mediated protection against SIV. Nature medicine, 24(10), 1590-1598.

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Assessing NK Cell Activation by gp120

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An assay to determine the expression of surface CD107a and intracellular production of IFN-γ and MIP-1β was performed by pulsing the CEM-NKr CCR5+ T lymphoblast cell line with gp120 SF162 (60 μg/ml), as previously described [16 ]. NK cells were isolated from whole blood from healthy donors using negative selection with RosetteSep (STEMCELL Technologies), then combined with CEM-NKr cells at a ratio of 5:1. Purified IgG, anti–CD107a–phycoerythrin (PE)–Cy5 (BD), brefeldin A (10 mg/ml) (Sigma), and GolgiStop (BD) were added for 5 hours at 37°C. The cells were then first stained for surface markers using anti–CD16–allophycocyanin (APC)–Cy7 (BD), anti–CD56-PE-Cy7 (BD), and anti–CD3–Alexa Fluor 700 (BD) and then stained intracellularly with anti–IFN-γ–APC (BD) and anti–MIP-1β–PE (BD) using Fix and Perm A and B solutions (Invitrogen). The cells were then fixed in 4% paraformaldehyde and analyzed by flow cytometry. NK cells were defined as CD3-negative and CD16- and/or CD56-positive.

Ackerman M.E., Mikhailova A., Brown E.P., Dowell K.G., Walker B.D., Bailey-Kellogg C., Suscovich T.J, & Alter G. (2016). Polyfunctional HIV-Specific Antibody Responses Are Associated with Spontaneous HIV Control. PLoS Pathogens, 12(1), e1005315.

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ELISA-Based NK Cell Activation Assay

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ELISA-based antibody-dependent NK cell activation assays were performed^{18 (link),62 (link)}. ELISA plates (Thermo Fisher NUNC MaxiSorp flat bottom) were coated with PPD (300 ng/well) or BSA as a negative control at 4°C for 16 hrs. Plasma (at 1:100, 1:1000, 1:10,000 dilutions in PBS) was added to each well. NK cells were isolated from whole blood from healthy HIV negative donors with RosetteSep (Stem Cell Technologies). NK cells (5x10⁴ per well), anti-CD107a-phycoerythrin (PE)-Cy5 (BD), brefeldin A (10 mg/ml) (Sigma), and GolgiStop (BD) were added and incubated for 5 hrs at 37°C. Cells were stained for surface markers using anti-CD16–allophycocyanin (APC)-Cy7 (BD), anti-CD56-PE-Cy7 (BD), and anti-CD3-AlexaFluor 700 (BD), and intracellularly with anti-IFNγ-APC (BD) and anti-MIP1β-PE (BD) using Fix and Perm A and B solutions (ThermoFisher). NK cells were defined as CD3- and CD16/56+ (Extended data Figure 8C). NK cell activation assays were performed in across dilutions stated above using cells from four healthy HIV negative donors.

Lu L.L., Smith M.T., Yu K.K., Luedemann C., Suscovich T.J., Grace P.S., Cain A., Yu W.H., McKitrick T.R., Lauffenburger D., Cummings R.D., Mayanja-Kizza H., Hawn T.R., Boom W.H., Stein C.M., Fortune S.M., Seshadri C, & Alter G. (2019). IFN-γ-independent immune markers of Mycobacterium tuberculosis exposure. Nature Medicine, 25(6), 977-987.

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NK Cell Activation Assay Protocol

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An assay to determine NK cell activation (NKA) state based on the expression of surface CD107a and intracellular production of IFNγ and MIP1β was performed as previously described. NK cells were isolated from whole blood from healthy donors using negative selection with RosetteSep (STEMCELL Technologies). Following a pulse with SF162 gp120 (60 μg/ml), T lymphoblast CEM‐NKr cells and isolated primary NK cells were mixed at a ratio of 1:5, and purified IgG, anti‐CD107a‐phycoerythrin (PE)‐Cy5 (BD), brefeldin A (10 mg/ml) (Sigma), and GolgiStop (BD) were added. After a 5‐h incubation at 37°C, cells were first stained for surface markers using anti‐CD16‐allophycocyanin (APC)‐Cy7 (BD), anti‐CD56‐PE‐Cy7 (BD), and anti‐CD3‐Alexa Fluor 700 (BD) and then stained intracellularly with anti‐IFNγ‐APC (BD) and anti‐MIP1β‐PE (BD) after treatment with Fix and Perm A and B solutions (Invitrogen). Cells were then fixed in 4% paraformaldehyde and analyzed by flow cytometry. NK cells were defined as CD3‐negative and CD16‐positive and/or CD56‐positive, and the percent of NK cells positive for each marker was determined.

Alter G., Dowell K.G., Brown E.P., Suscovich T.J., Mikhailova A., Mahan A.E., Walker B.D., Nimmerjahn F., Bailey‐Kellogg C, & Ackerman M.E. (2018). High‐resolution definition of humoral immune response correlates of effective immunity against HIV. Molecular Systems Biology, 14(3), e7881.

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NK Cell-Mediated Immune Function Assay

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An assay to determine the expression of surface CD 107a and intracellular production of IFN-γ and MIP-1β was performed by pulsing the CEM-NKr CCR5⁺ T lymphoblast cell line with rgp120SIV_mac251 (60 mg/ml), as previously described^{51 (link)}. NK cells were isolated from whole blood from healthy donors using negative selection with RosetteSep (STEMCELL Technologies), as recommended by the manufacturer. The CEM-NKr cells and isolated primary NK cells were mixed at a ratio of 1:5, and purified IgG, anti- CD107a-phycoerythrin (PE)-Cy5 (BD), brefeldin A (10 mg/ml) (Sigma-Aldrich), and GolgiStop (BD) were added for 5 h at 37 °C. The cells were then first stained for surface markers using anti-CD 16-allophycocyanin (APC)-Cy7 (BD), anti-CD56-PE-Cy7 (BD) and anti-CD3-Alexa Fluor 700 (BD) and then stained intracellularly with anti-IFN-γ-APC (BD) and anti-MIP-1β-PE (BD) using Fix and Perm A and B solutions (Invitrogen). The cells were then fixed in 4% paraformaldehyde and analyzed using flow cytometry. NK cells were defined as CD3⁻ and CD16⁻ and/or CD56⁺.

Vaccari M., Gordon S.N., Fourati S., Schifanella L., Liyanage N.P., Cameron M., Keele B.F., Shen X., Tomaras G.D., Billings E., Rao M., Chung A.W., Dowell K.G., Bailey-Kellogg C., Brown E.P., Ackerman M.E., Vargas-Inchaustegui D.A., Whitney S., Doster M.N., Binello N., Pegu P., Montefiori D.C., Foulds K., Quinn D.S., Donaldson M., Liang F., Loré K., Roederer M., Koup R.A., McDermott A., Ma Z.M., Miller C.J., Phan T.B., Forthal D.N., Blackburn M., Caccuri F., Bissa M., Ferrari G., Kalyanaraman V., Ferrari M.G., Thompson D., Robert-Guroff M., Ratto-Kim S., Kim J.H., Michael N.L., Phogat S., Barnett S.W., Tartaglia J., Venzon D., Stablein D.M., Alter G., Sekaly R.P, & Franchini G. (2016). Adjuvant-dependent innate and adaptive immune signatures of risk of SIVmac251 acquisition. Nature medicine, 22(7), 762-770.

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NK Cell Functional Assay by ELISA

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ELISA plates were coated with PPD (300 ng/well) or BSA at 4°C for 16 hours [33 (link)]. Purified IgG (25 µg) from study participants was added to each well. NK cells were isolated from whole blood from seronegative donors with RosetteSep. NK cells (5 × 10⁴/well) were incubated with anti-CD107a–phycoerythrin (PE)–Cy5 (BD), brefeldin A (10 mg/mL) (Sigma), and GolgiStop (BD) at 37°C for 5 hours. Cells were stained for surface markers using anti-CD16–allophycocyanin (APC)–Cy7 (BD), anti-CD56–PE–Cy7 (BD), and anti-CD3–AlexaFluor 700 (BD), and then intracellularly with anti-IFN-γ–APC (BD) and anti-MIP1β–PE (BD) using Fix and Perm A and B solutions (ThermoFisher). Frequency (%) of NK cells positive for CD107a, IFN-γ, and MIP1β were determined with NK cells defined as CD3⁻ and CD16/56⁺.

Lu L.L., Das J., Grace P.S., Fortune S.M., Restrepo B.I, & Alter G. (2020). Antibody Fc Glycosylation Discriminates Between Latent and Active Tuberculosis. The Journal of Infectious Diseases, 222(12), 2093-2102.

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ELISA-based NK cell activation assay

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ELISA-based Ab-dependent NK cell activation assay was modified for use with ID93 antigen.^{24 (link)} Briefly, ELISA plates (Thermo Fisher NUNC MaxiSorp flat bottom) were coated with ID93 (3 ng/well) or BSA as a negative control at 4 °C for 16 h. Serum samples from Days 0 and 84 from all cohorts were diluted 1:100 and added to each well. NK cells were isolated from whole blood from HIV-seronegative donors with RosetteSep (STEMCELL Technologies) and cultured overnight with IL-15 (1 ng/mL). NK cells (5 × 10⁴ per well), anti-CD107a-phycoerythrin (PE)-Cy5 (catalog number 555801, BD), brefeldin A (10 mg/mL Sigma), and GolgiStop (BD) were added to each well, and the plates were incubated for 5 h at 37 °C. Cells were then stained for surface markers using anti-CD16–allophycocyanin (APC)-Cy7 (catalog number 557758, BD), anti-CD56-PE-Cy7 (catalog number 557747, BD), and anti-CD3-AlexaFluor 700 (catalog number 557943, BD), and then stained intracellularly with anti-IFNγ-APC (catalog number 554702, BD) and anti-MIP1β-PE (catalog number 550078, BD) using Fix and Perm A and B solutions (Thermo Fisher Scientific). Fixed cells were analyzed by flow cytometry. NK cells were defined as CD3− and CD16/56+.

Coler R.N., Day T.A., Ellis R., Piazza F.M., Beckmann A.M., Vergara J., Rolf T., Lu L., Alter G., Hokey D., Jayashankar L., Walker R., Snowden M.A., Evans T., Ginsberg A, & Reed S.G. (2018). The TLR-4 agonist adjuvant, GLA-SE, improves magnitude and quality of immune responses elicited by the ID93 tuberculosis vaccine: first-in-human trial. NPJ Vaccines, 3, 34.

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Antibody-Dependent NK Cell Degranulation Assay

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Antibody dependent NK cell degranulation as described previously (49). Briefly, SARS-CoV-2 antigens were coated to 96-well ELISA at the protein concentration of 2 g/ml, incubated at 37°C for 2hrs and blocked with 5% BSA at 4°C overnight. NK cells were isolated from whole blood from healthy donors (by negative selection using RosetteSep (STEMCELL) then separated using a ficoll gradient. NK cells were rested overnight in media supplemented with IL-15. Serum samples were diluted at 1:25. After blocking, samples were added to coated plates and immune complexes were formed for two hours at 37°C. After the two hours, NK cells were prepared (antiCD107a– phycoerythrin (PE) – Cy5 (BD), brefeldin A (10 μg/ml) (Sigma), and GolgiStop (BD)), and added to each well. for 5 hours at 37°C. The cells were stained for surface markers using anti-CD3 PacBlue (BD), anti-CD16 APC-Cy5 (BD), and anti-CD56 PE-Cy7 (BD) and permeabilized. The flow cytometry was performed. NK cells were gates as CD3−, CD16+, CD56+ cells and NK cell activity was determined as the percent of NK cells positive for CD107a and MIP-1b.

Kaplonek P., Cizmeci D., Fischinger S., Collier A.R., Suscovich T., Linde C., Broge T., Mann C., Amanat F., Dayal D., Rhee J., de St. Aubin M., Nilles E.J., Musk E.R., Menon A.S., Saphire E.O., Krammer F., Lauffenburger D.A., Barouch D.H, & Alter G. (2021). Subtle immunological differences in mRNA-1273 and BNT162b2 COVID-19 vaccine induced Fc-functional profiles. bioRxiv.

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