Intracellular expression of the heavy and light chains of antibody molecules was determined by staining cells with fluorescently‐labeled antibodies specific for heavy or light chains (Roy, Miro‐Quesada, et al., 2017 ). Briefly, the cells were centrifuged, washed with FACS buffer and fixed with Fixation Medium A (Life Technologies) for 15 min at room temperature. Next, the cells were washed with FACS buffer and stained for 15 min at room temperature with the staining solution comprised of goat anti‐human IgG (Fc)‐Alexa Fluor(AF)488 (Life Technologies) and goat anti‐human κ‐APC (Biolegend) in Permeabilization Medium B (Life Technologies). The stained cells were washed and resuspended in FACS buffer before analysis on an LSRII for the APC and AF488 double‐positive population. Data analysis was performed using FlowJo software.
Permeabilization medium b
Manufactured by Thermo Fisher Scientific
Permeabilization Medium B is a laboratory reagent used to permeabilize cells, allowing for the introduction of substances into the cell interior. It is a specialized buffer solution designed to temporarily disrupt the cell membrane without compromising cell viability. The core function of Permeabilization Medium B is to facilitate the delivery of molecules, such as antibodies, proteins, or genetic material, into the intracellular environment for various research and analytical applications.
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Lab products found in correlation
Fixation medium a, by Thermo Fisher Scientific (2 mentions)
Ifn γ apc, by BD (1 mentions)
Cd16 apc cy7, by BD (1 mentions)
Cd3 alexa fluor 700, by BD (1 mentions)
Cd107a pe cy5, by BD (1 mentions)
Cd56 pe cy7, by BD (1 mentions)
Mip1β pe, by BD (1 mentions)
Brefeldin a, by Merck Group (1 mentions)
Golgistop, by BD (1 mentions)
2 protocols using permeabilization medium b
1
Antibody Intracellular Expression Profiling
2
Phenotyping NK Cell Activation
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Enzyme-linked immunosorbent assay plates were coated with measles antigen and incubated with samples diluted 1:20, or PBS as negative control, for 2 hours at 37°C. Natural killer cells were isolated from buffy coats collected from blood bank donors. CD107a-PE/Cy5 (BD), brefeldin A (Sigma), GolgiStop (BD), and NK cells were added to the antigen-coated plate and incubated for 5 hours at 37°C. Next, cells were transferred to a V-bottom plate and stained with CD56-PE/Cy7, CD16-APC/Cy7, and CD3-AlexaFluor700 (all BD) for 15 minutes at room temperature. Cells were washed and fixed in Fixation Medium A (Life Technologies) before intracellular staining with MIP-1β-PE and IFN-γ-APC (BD) in Permeabilization Medium B (Life Technologies) for 15 minutes at room temperature. The percentages of positive NK cells for CD107a, IFN-γ, and MIP-1β were determined by flow cytometry.
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