legendplex human cytokine detection kit  (Revvity)

Journal: STAR Protocols

Article Title: A protocol for high-throughput screening for immunomodulatory compounds using human primary cells

doi: 10.1016/j.xpro.2023.102405

Figure Lengend Snippet:

Article Snippet: IL-10 AlphaLISA Detection Kit, 5,000 Assay Points , PerkinElmer , Product Number: AL218F.

Techniques: Recombinant, Sterility, Modification, Staining, Saline, Multiplex Assay, Software

Journal: STAR Protocols

Article Title: A protocol for high-throughput screening for immunomodulatory compounds using human primary cells

doi: 10.1016/j.xpro.2023.102405

Figure Lengend Snippet: Representative cytokine induction results (A) Raw luminescence values (RLU) of a TNF AlphaLISA assay are depicted. Color gradients from white to red indicate lower to higher values. The negative and positive controls for the AlphaLISA assay were DMSO and R848, respectively. (B) Mean raw luminescence values plotted as bar plots with errors shown as SEM. Each group was compared by one way ANOVA. For statistical analyses: ns p > 0.05, ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001, ∗∗∗∗p ≤ 0.0001.

Article Snippet: IFN-γ AlphaLISA Detection Kit, 5,000 Assay Points , PerkinElmer , Product Number: AL217F.

Techniques:

Journal: STAR Protocols

Article Title: A protocol for high-throughput screening for immunomodulatory compounds using human primary cells

doi: 10.1016/j.xpro.2023.102405

Figure Lengend Snippet: Representative schematic of the AlphaLISA assay system A total of 8 μL of acceptor bead solution, containing biotinylated antibodies as well as acceptor beads, were added to each well containing 2 μL of supernatant. After a 1 h incubation, a 10 μL solution of Streptavidin donor beads were added to this solution and incubated for 30 min. Both beads are manufactured to be specific to a binding site for the cytokine of interest. When read on the EnVision Plate Reader, bound donor and acceptor beads, due to their proximity, will emit a 615 nm luminescence when excited by a 680 nm wavelength. The intensity of this luminescence reflects the concentration of bound beads or the concentration of the cytokine.

Article Snippet: IFN-γ AlphaLISA Detection Kit, 5,000 Assay Points , PerkinElmer , Product Number: AL217F.

Techniques: Incubation, Binding Assay, Concentration Assay

Journal: STAR Protocols

Article Title: A protocol for high-throughput screening for immunomodulatory compounds using human primary cells

doi: 10.1016/j.xpro.2023.102405

Figure Lengend Snippet: RPMI and DMEM as cell culture media options for screening platform Heat map and density distribution of luminescence readouts for a representative TNF AlphaLISA readout using DMEM (left) and RPMI (right). The trace amounts of biotin in RPMI causes high levels of interference and induces artificially high luminescence readouts in many wells.

Article Snippet: IFN-γ AlphaLISA Detection Kit, 5,000 Assay Points , PerkinElmer , Product Number: AL217F.

Techniques: Cell Culture

Journal: STAR Protocols

Article Title: A protocol for high-throughput screening for immunomodulatory compounds using human primary cells

doi: 10.1016/j.xpro.2023.102405

Figure Lengend Snippet: Antibody cocktail and AlphaLISA assay materials

Article Snippet: IFN-γ AlphaLISA Detection Kit, 5,000 Assay Points , PerkinElmer , Product Number: AL217F.

Techniques: Concentration Assay

Journal: STAR Protocols

Article Title: A protocol for high-throughput screening for immunomodulatory compounds using human primary cells

doi: 10.1016/j.xpro.2023.102405

Figure Lengend Snippet: Representative cytokine induction results (A) Raw luminescence values (RLU) of a TNF AlphaLISA assay are depicted. Color gradients from white to red indicate lower to higher values. The negative and positive controls for the AlphaLISA assay were DMSO and R848, respectively. (B) Mean raw luminescence values plotted as bar plots with errors shown as SEM. Each group was compared by one way ANOVA. For statistical analyses: ns p > 0.05, ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001, ∗∗∗∗p ≤ 0.0001.

Article Snippet: TNF-α AlphaLISA Detection Kit, 5,000 Assay Points , PerkinElmer , Product Number: AL208F.

Techniques:

Journal: STAR Protocols

Article Title: A protocol for high-throughput screening for immunomodulatory compounds using human primary cells

doi: 10.1016/j.xpro.2023.102405

Figure Lengend Snippet: Representative schematic of the AlphaLISA assay system A total of 8 μL of acceptor bead solution, containing biotinylated antibodies as well as acceptor beads, were added to each well containing 2 μL of supernatant. After a 1 h incubation, a 10 μL solution of Streptavidin donor beads were added to this solution and incubated for 30 min. Both beads are manufactured to be specific to a binding site for the cytokine of interest. When read on the EnVision Plate Reader, bound donor and acceptor beads, due to their proximity, will emit a 615 nm luminescence when excited by a 680 nm wavelength. The intensity of this luminescence reflects the concentration of bound beads or the concentration of the cytokine.

Article Snippet: TNF-α AlphaLISA Detection Kit, 5,000 Assay Points , PerkinElmer , Product Number: AL208F.

Techniques: Incubation, Binding Assay, Concentration Assay

Journal: STAR Protocols

Article Title: A protocol for high-throughput screening for immunomodulatory compounds using human primary cells

doi: 10.1016/j.xpro.2023.102405

Figure Lengend Snippet: RPMI and DMEM as cell culture media options for screening platform Heat map and density distribution of luminescence readouts for a representative TNF AlphaLISA readout using DMEM (left) and RPMI (right). The trace amounts of biotin in RPMI causes high levels of interference and induces artificially high luminescence readouts in many wells.

Article Snippet: TNF-α AlphaLISA Detection Kit, 5,000 Assay Points , PerkinElmer , Product Number: AL208F.

Techniques: Cell Culture

Journal: STAR Protocols

Article Title: A protocol for high-throughput screening for immunomodulatory compounds using human primary cells

doi: 10.1016/j.xpro.2023.102405

Figure Lengend Snippet: Antibody cocktail and AlphaLISA assay materials

Article Snippet: TNF-α AlphaLISA Detection Kit, 5,000 Assay Points , PerkinElmer , Product Number: AL208F.

Techniques: Concentration Assay

Journal: bioRxiv

Article Title: Tolerance to Haemophilus influenzae infection in human epithelial cells: insights from a primary cell-based model

doi: 10.1101/2023.07.11.548529

Figure Lengend Snippet: Prepared with BioRender.com. Panel B: Bacterial loads during infection. Total bacterial and intracellular bacterial loads (CFU/mL) were determined on Day 1, Day 3, Day 7 and Day 14. Each dot represents the average CFU/mL observed for each donor ( n =2 biological replicates for each donor and condition). Bars and error bars indicate average CFU/mL and standard error. Inoc is the CFU/mL in the bacterial inoculum used to infect NHNE. Panel C: Changes in LDH-release in infected NHNE. LDH levels in infected and uninfected samples were determined using AlphaLISA. The fold change in infected compared to uninfected NHNE from each donor is shown, averages and standard error are shown. Panel D: Transepithelial resistance (TEER) changes resulting from NHNE infection . TEER readings for uninfected and infected NHNE for all donors are shown. Due to instrument failure, no readings were available for Donor 1 samples on Day 3 of infection. Average reading and standard error are shown. Panel E: Immunofluorescence Microscopy of infected NHNE. Representative images show donor 3 samples. White – Phalloidin stand of filamentous actin; Blue – cellular nuclei, DAPI stain; Green – TUNEL labelling; Red – anti-NTHi antibody. In Panels A, B &C, data plotted for each timepoint represent the average of multiple biological replicate measurements for samples from an individual donor.

Article Snippet: Apical wash fractions collected pre- and post-infection were used to detect Lactate dehydrogenase activity and IL-8 concentrations using human LDH and IL-8 Alphalisa Kits (Perkin Elmer).

Techniques: Infection, Immunofluorescence, Microscopy, Staining, TUNEL Assay