Anti cd107a af488 antibody

Manufactured by BioLegend

Sourced in United States

The Anti-CD107a-AF488 antibody is a fluorescently labeled monoclonal antibody that specifically binds to the CD107a (LAMP-1) protein. CD107a is a marker of lysosomal-associated membrane protein that is expressed on the cell surface during degranulation, a process that occurs in activated immune cells such as natural killer cells and cytotoxic T cells.

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Anti-CD107a antibody Anti-CD107a

5 protocols using anti cd107a af488 antibody

Evaluating NK Cell Activity Assay

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The ADCC assay for measuring intracellular NK cell IFN-γ and CD107a expression was conducted and analysed with the gating strategy as previously described^{23 (link)}. Briefly, 96-well plates were coated overnight at 4 °C with A/California/04/09 HA protein (1 μg/ml) and chimeric cH9/1 HA protein (1 μg/ml). The plates were then washed with PBS and incubated with heat-inactivated sera (prediluted 1:10) for 2 h at 37 °C. Plates were then washed again with PBS and incubated with 10⁵ CD16 176 v NK-92 cells per well (mycoplasma-free, human NK cell line expressing high affinity 176 V variant CD16 receptor) (Fox Chase Cancer Center, Philadelphia, PA, USA). As a negative control, NK-92 cells lacking the expression of CD16 were added to an additional well per sample. Cells were incubated with anti-CD107a-AF488 antibody (Biolegend, San Diego, CA, USA), Brefeldin A (5 μg/ml, BD) and monensin (5 μg/ml, BD) for 16 h at 37 °C. After incubation, the cells were stained with LIVE/DEAD Fixable Aqua dead cell staining kit (Invitrogen), anti-CD3-PE CF594 (BD) and anti-CD56-APC (BD) before intracellular staining with anti-IFN-γ-BV-421 (Biolegend). The cells were acquired on BD Fortessa. Data analysis was performed using FlowJo version 10 (treeStar).

Madsen A., Jul-Larsen Å., Trieu M.C., Krammer F, & Cox R.J. (2021). Persistently high antibody responses after AS03-adjuvanted H1N1pdm09 vaccine: Dissecting the HA specific antibody response. NPJ Vaccines, 6, 45.

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ADCC Assay for NK Cell Function

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The ADCC assay measuring intracellular NK cell IFNγ and CD107a expression was conducted as previously described with minor modifications.^{35 (link)} Briefly, 96-well plates were coated overnight at 4 °C with 1 μg/ml HA1 (A/California/06/2009(H1N1)) 6 × His tagged (eEnzyme, USA) or chimeric cH6/1 in PBS. Plates were washed with PBS and incubated with heat-inactivated human sera for 2 h at 37 °C. After washing, 10⁵ CD16 176v NK-92 cells (mycoplasma-free, human NK cell line expressing high affinity 176V variant CD16 receptor) (kindly provided by Fox Chase Cancer Center, Philadelphia, PA, USA) were added per test well. As a negative control for each sample, NK-92 cells (lacking expression of CD16) were added to an additional well. The cells were incubated for 16 h at 37 °C with anti-CD107a-AF488 antibody (Biolegend, San Diego, CA, USA, 328610), Brefeldin A (5 μg/ml, BD) and monensin (5 μg/ml, BD). Cells were stained with LIVE/DEAD Fixable Aqua dead cell staining kit (Invitrogen, Carlsbad, CA, USA), anti-CD3-PE CF594 (BD, Franklin Lakes, NJ, USA, 562280) and anti-CD56-APC (BD, 555518) before intracellular staining with anti-IFN-γ-BV-421 (Biolegend, 502532). Cells were acquired on BD Fortessa (San Jose, CA, USA). Data analysis was done using FlowJo version 10 (treeStar, Ashland, OR, USA).

Tete S.M., Krammer F., Lartey S., Bredholt G., Wood J., Skrede S, & Cox R.J. (2016). Dissecting the hemagglutinin head and stalk-specific IgG antibody response in healthcare workers following pandemic H1N1 vaccination. NPJ Vaccines, 1, 16001-.

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NK Cell-Mediated ADCC Assay

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The ADCC assay measuring intracellular NK cell IFNγ and CD107a expression was conducted as previously described with minor modifications.^{35 (link)} Briefly, 96-well plates were coated overnight at 4 °C with 1 μg/ml HA1 (A/California/06/2009(H1N1)) 6×His tagged (eEnzyme, USA) or chimeric cH6/1 in PBS. Plates were washed with PBS and incubated with heat-inactivated human sera for 2 h at 37 °C. After washing, 10⁵ CD16 176v NK-92 cells (mycoplasma-free, human NK cell line expressing high affinity 176V variant CD16 receptor) (kindly provided by Fox Chase Cancer Center, Philadelphia, PA, USA) were added per test well. As a negative control for each sample, NK-92 cells (lacking expression of CD16) were added to an additional well. The cells were incubated for 16 h at 37 °C with anti-CD107a-AF488 antibody (Biolegend, San Diego, CA, USA, 328610), Brefeldin A (5 μg/ml, BD) and monensin (5 μg/ml, BD). Cells were stained with LIVE/DEAD Fixable Aqua dead cell staining kit (Invitrogen, Carlsbad, CA, USA), anti-CD3-PE CF594 (BD, Franklin Lakes, NJ, USA, 562280) and anti-CD56-APC (BD, 555518) before intracellular staining with anti-IFN-γ-BV-421 (Biolegend, 502532). Cells were acquired on BD Fortessa (San Jose, CA, USA). Data analysis was done using FlowJo version 10 (treeStar, Ashland, OR, USA).

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ADCC Assay for NK Cell Activity

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ADCC assay for intracellular natural killer (NK) cell interferon-gamma (IFNγ) and CD107a expression was conducted as previously described, and analyzed with the same gating strategy [10] (link). Briefly, 96-well plates were coated overnight at 4 °C with chimeric cH9/1 HA protein (1 μg/ml). The next day, plates were washed with PBS and incubated with heat-inactivated sera (sera dilution 1:10) for two hours at 37 °C. Plates were washed again with PBS and incubated with 10⁵ CD16 176v NK-92 cells per well (mycoplasma-free, human NK cell line expressing high affinity 176 V variant CD16 receptor) (Fox Chase Cancer Center, Philadelphia, PA, USA). As a negative control, NK-92 cells lacking expression of CD16 were added to an additional well for each sample.
The cells were incubated with anti-CD107a-AF488 antibody (Biolegend, San Diego, CA, USA), Brefeldin A (5 μg/ml, BD) and monensin (5 μg/ml, BD) for 16 h at 37 °C. After incubation, cells were stained with LIVE/DEAD Fixable Aqua dead cell staining kit (Invitrogen), anti-CD3-PE CF594 (BD) and anti-CD56-APC (BD) before intracellular staining with anti-IFN-γ-BV-421 (Biolegend). Cells were acquired on BD Fortessa. Data analysis was done using FlowJo version 10 (treeStar).

Madsen A., Azimi L., Tete S., Zhou F., Krammer F., Cox R.J, & Jul-Larsen Å. (2019). No evidence of antigenic seniority in hemagglutinin specific antibody responses after adjuvanted pandemic 2009 influenza vaccination. Vaccine: X, 2, 100029.

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Multiparametric Analysis of Tumor-Infiltrating T Cells

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Thawed live cells were stained with and anti-CD103 BV421, anti-CD45 BV510, anti-CD8 BV605, anti-CCR7 BV711, Anti-CD45Ro BV785, anti-CD69 PEcy7, anti-CD4 AF700 and anti-CD19 APC-cy7, anti-CD11c APC/Fire 750 (all from Biolegend), prior to sorting as above. Live melanoma cells (CD45^- SSC-A^high FSC-A^high cells) and three populations of CD8⁺ CD4^- T cells were sorted, the latter as live CD11c^- CD19^- CD45⁺ CD45Ro⁺ CCR7^- lymphocytes distinguished by the marker combination CD69⁺ CD103^-, CD69⁺ CD103⁺ and CD69^- CD103^-.
T cells were stained with anti-PD1(pembrolizumab) or Isotype IgG₄ (BD). T cells and melanoma cells were cultured for 24 hours at a 2:1 effector to target ratio with anti-CD107a-AF488 antibody (Biolegend) or BD GolgiPlug (Brefeldin A) in complete human media (online supplemental methods). For non-sorted samples, 50,000 cells from tumor samples or 25,000 cells from expanded TILs were cocultured.
After 24 hours, cells were restained with all the extracellular markers used for sorting, adding anti-41BB-PercP-Cy5.5 (Biolegend) to anti-CD107a stained cells. Cells incubated with GolgiPlug were permeabilized with eBioscience FoxP3/Transcription Factor Staining Buffer Set (Invitrogen) prior to intracellular staining with anti-IFNγ-PEDazzle594, anti-TNFα-APC, anti-Granzyme B-FITC and IL-2-PercPCy5.5 (all Biolegend).

Pizzolla A., Keam S.P., Vergara I.A., Caramia F., Thio N., Wang M., Kocovski N., Tantalo D., Jabbari J., Au-Yeung G., Sandhu S., Gyorki D.E., Weppler A., Perdicchio M., McArthur G.A., Papenfuss A.T, & Neeson P.J. (2022). Tissue-resident memory T cells from a metastatic vaginal melanoma patient are tumor-responsive T cells and increase after anti-PD-1 treatment. Journal for Immunotherapy of Cancer, 10(5), e004574.

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