Journal: bioRxiv

Article Title: Chronic obstructive pulmonary disease and cigarette smoke exposure lead to dysregulated MAIT cell activation by bronchial epithelial cells

doi: 10.1101/2022.02.28.482383

Figure Lengend Snippet: a) Primary BEC from healthy (n=7), COPD (n=6), or smoker (n=6) donors were incubated with the D426 G11 MAIT cell clone in an ELISPOT assay with IFN-γ production as the readout. Data points are the mean IFN-γ spot-forming units (SFU) of two technical replicates per donor. Statistical analysis was performed as described in the experimental procedures and is summarized in . b-c) BEC from a representative healthy and COPD donor were incubated with blocking antibodies to IL-12/IL-18 or MR1 five hours prior to addition of the MAIT cells in an IFN-γ ELISPOT assay. Results are presented as b) the mean of two experimental replicates and c) representative ELISPOT well images.

Article Snippet: The following antibodies were used: for ELISPOT assays: α-MR1 (26.5, Biolegend), α-IL-12p70 (MAB219100, R&D systems), α-IL-18 (D044-3, MBL International Corporation), α-IgG2A isotype (400224, Biolegend), α-human IFN-γ (7-B6-1, MabTech); for fluorescence microscopy: α-human HLA-A,B,C (W6/32, biotinylated, Biolegend), streptavidin-AlexaFluor-647 (Life Technologies); for flow cytometry: α-MR1 (26.5, conjugated to APC, Biolegend), α-human HLA-A,B,C (W6/32, conjugated to APC, Biolegend).

Techniques: Incubation, Enzyme-linked Immunospot, Blocking Assay

Journal: bioRxiv

Article Title: Chronic obstructive pulmonary disease and cigarette smoke exposure lead to dysregulated MAIT cell activation by bronchial epithelial cells

doi: 10.1101/2022.02.28.482383

Figure Lengend Snippet: a) Primary BEC from healthy, COPD, or smoker donors were infected with media control, M. smegmatis (0.1μl/well), or S. pneumoniae (MOI 20) for one hour prior to addition of MAIT cells in an IFN-γ ELISPOT assay. Data points are the mean IFN-γ spot-forming units (SFU) of two technical replicates per donor. Statistical analysis was performed as described in the experimental procedures and is summarized in . b) BEC from a representative healthy and COPD donor were incubated with blocking antibodies to IL-12/IL-18 or MR1 one hour prior to infection with S. pneumoniae (MOI 20) and subsequent addition of the MAIT cell clones in an IFN-γ ELISPOT assay. Results are presented as the mean of two experimental replicates.

Article Snippet: The following antibodies were used: for ELISPOT assays: α-MR1 (26.5, Biolegend), α-IL-12p70 (MAB219100, R&D systems), α-IL-18 (D044-3, MBL International Corporation), α-IgG2A isotype (400224, Biolegend), α-human IFN-γ (7-B6-1, MabTech); for fluorescence microscopy: α-human HLA-A,B,C (W6/32, biotinylated, Biolegend), streptavidin-AlexaFluor-647 (Life Technologies); for flow cytometry: α-MR1 (26.5, conjugated to APC, Biolegend), α-human HLA-A,B,C (W6/32, conjugated to APC, Biolegend).

Techniques: Infection, Control, Enzyme-linked Immunospot, Incubation, Blocking Assay, Clone Assay

Journal: bioRxiv

Article Title: Chronic obstructive pulmonary disease and cigarette smoke exposure lead to dysregulated MAIT cell activation by bronchial epithelial cells

doi: 10.1101/2022.02.28.482383

Figure Lengend Snippet: a-b) BEC from representative healthy, COPD, or smoker donors were infected with fluorescently labeled S. pneumoniae for three hours. Fixed cells were stained with DAPI and α-MHC-Ia antibody to label the cell surface. Approximately 20 fields per donor were selected without bias based on nuclear stain, and whole cells within these fields were then analyzed by Imaris to enumerate the number of bacteria associated with individual cells. a) Data points indicate individual cells, analyzed by one-way ANOVA statistical analysis. b) Representative images of S. pneumoniae -infected primary BEC. White = MHC-Ia surface staining. Red pseudocolor = fluorescent S. pneumoniae . Arrows indicate adherent S. pneumoniae (yellow) enumerated for analysis. c) IFN-γ SFU fold change between no-treatment control and M. smegmatis- or S. pneumoniae- infected primary BEC from healthy, COPD, or smoker donors. Raw data shown in Figure 2a. Statistical analysis was performed as described in the experimental procedures and is summarized in .

Article Snippet: The following antibodies were used: for ELISPOT assays: α-MR1 (26.5, Biolegend), α-IL-12p70 (MAB219100, R&D systems), α-IL-18 (D044-3, MBL International Corporation), α-IgG2A isotype (400224, Biolegend), α-human IFN-γ (7-B6-1, MabTech); for fluorescence microscopy: α-human HLA-A,B,C (W6/32, biotinylated, Biolegend), streptavidin-AlexaFluor-647 (Life Technologies); for flow cytometry: α-MR1 (26.5, conjugated to APC, Biolegend), α-human HLA-A,B,C (W6/32, conjugated to APC, Biolegend).

Techniques: Infection, Labeling, Staining, Bacteria, Control

Journal: bioRxiv

Article Title: Chronic obstructive pulmonary disease and cigarette smoke exposure lead to dysregulated MAIT cell activation by bronchial epithelial cells

doi: 10.1101/2022.02.28.482383

Figure Lengend Snippet: a-b) Primary BEC from healthy (n=7), COPD (n=6), or smoker (n=6) donors were incubated with media containing 0% or 30% CSE for three hours prior to the addition of MAIT cells in an IFN-γ ELISPOT assay. Statistical analysis was performed as described in the experimental procedures and is summarized in - . a) Data points are the mean IFN-γ spot-forming units (SFU) of two technical replicates, paired by individual donor. b) IFN-γ SFU fold change between 0% CSE- and 30% CSE-treated primary BEC from healthy, COPD, or smoker donors, calculated pairwise by donor.

Article Snippet: The following antibodies were used: for ELISPOT assays: α-MR1 (26.5, Biolegend), α-IL-12p70 (MAB219100, R&D systems), α-IL-18 (D044-3, MBL International Corporation), α-IgG2A isotype (400224, Biolegend), α-human IFN-γ (7-B6-1, MabTech); for fluorescence microscopy: α-human HLA-A,B,C (W6/32, biotinylated, Biolegend), streptavidin-AlexaFluor-647 (Life Technologies); for flow cytometry: α-MR1 (26.5, conjugated to APC, Biolegend), α-human HLA-A,B,C (W6/32, conjugated to APC, Biolegend).

Techniques: Incubation, Enzyme-linked Immunospot

Journal: bioRxiv

Article Title: Chronic obstructive pulmonary disease and cigarette smoke exposure lead to dysregulated MAIT cell activation by bronchial epithelial cells

doi: 10.1101/2022.02.28.482383

Figure Lengend Snippet: a-b) Primary BEC from healthy (n=7), COPD (n=6), or smoker (n=6) donors were incubated with media containing 0% or 30% CSE for three hours. BEC were infected with M. smegmatis (a, 0.05μl/well) or S. pneumoniae (b, 20 MOI) for one hour prior to the addition of MAIT cells in an IFN-γ ELISPOT assay. Statistical analysis was performed as described in the experimental procedures and is summarized in - . c) Fold change of a) and b) IFN-γ SFU between 0% CSE- and 30% CSE-treated primary BEC infected with M. smegmatis or S. pneumoniae , calculated pairwise by donor. d-e) BEC from representative healthy, COPD, or smoker donors were incubated with medium containing 0% or 30% CSE for three hours, then infected with fluorescently labeled S. pneumoniae for three hours. Fixed cells were stained with DAPI and α-MHC-Ia antibody to label the cell surface. Approximately 20 fields per donor were selected without bias based on nuclear stain, and whole cells within these fields were then analyzed by Imaris to enumerate the number of bacteria associated with individual cells. d) Representative images of S. pneumoniae -infected healthy BEC treated with 0% or 30% CSE. White = MHC-Ia surface staining. Red pseudocolor = fluorescent S. pneumoniae . Arrows indicate adherent S. pneumoniae (yellow) enumerated for analysis or extracellular microbes (red) excluded for analysis. e) Data points indicate individual cells, analyzed by one-way ANOVA statistical analysis. 0% CSE results from are included as reference.

Article Snippet: The following antibodies were used: for ELISPOT assays: α-MR1 (26.5, Biolegend), α-IL-12p70 (MAB219100, R&D systems), α-IL-18 (D044-3, MBL International Corporation), α-IgG2A isotype (400224, Biolegend), α-human IFN-γ (7-B6-1, MabTech); for fluorescence microscopy: α-human HLA-A,B,C (W6/32, biotinylated, Biolegend), streptavidin-AlexaFluor-647 (Life Technologies); for flow cytometry: α-MR1 (26.5, conjugated to APC, Biolegend), α-human HLA-A,B,C (W6/32, conjugated to APC, Biolegend).

Techniques: Incubation, Infection, Enzyme-linked Immunospot, Labeling, Staining, Bacteria

Journal: Vaccines

Article Title: Enhancement of Vaccine-Induced T-Cell Responses by Probiotics in Calves

doi: 10.3390/vaccines13111120

Figure Lengend Snippet: Effects of Zeosapo KB or CB feeding on T-cell subsets in PBMCs. ( A – F ) PBMCs isolated from calves (NC, circle, n = 20; Zeosapo KB, triangle, n = 19; CB, diamond, n = 18) 4 weeks after the booster vaccination were cultured only with medium for 5 days. The proportions (%) of CD25 + and CD25 + CD69 + cells in CD3 + CD4 + T cells ( A , B ) and CD3 + CD8 + T cells ( C , D ) were analyzed by flow cytometry. IFN-γ ( C ) and TNF-α ( D ) productions from PBMCs isolated from each animal were measured by ELISA in duplicate. Statistical significance was determined by the Dunn’s test after the Kruskal–Wallis test for multiple-group comparisons. * p < 0.05, ** p < 0.01, *** p < 0.001. ND, not detected. The data for IFN-γ and TNF-α included samples that were below the detection limit and the statistical analysis was not performed on these data.

Article Snippet: Culture supernatants of PBMCs were collected, and IFN-γ and TNF-α concentrations were measured using the Bovine IFN-γ ELISA Development Kit (Mabtech, Nacka Strand, Sweden) and Bovine TNF-α ELISA Development Kit (Mabtech), respectively, according to the manufacturers’ protocols.

Techniques: Isolation, Cell Culture, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Vaccines

Article Title: Enhancement of Vaccine-Induced T-Cell Responses by Probiotics in Calves

doi: 10.3390/vaccines13111120

Figure Lengend Snippet: Activation of Th1 cytokine responses to BRSV antigen. ( A , B ) PBMCs isolated from calves (NC, circle, n = 20; Zeosapo KB, triangle, n = 19; CB, diamond, n = 18) 4 weeks after the booster vaccination were cultured in the presence of BRSV antigen for 5 days. IFN-γ ( A ) and TNF-α ( B ) production from PBMCs isolated from each animal was measured by ELISA in duplicate. Statistical significance was determined using the Dunn’s test after the Kruskal–Wallis test for multiple-group comparisons. ** p < 0.01.

Article Snippet: Culture supernatants of PBMCs were collected, and IFN-γ and TNF-α concentrations were measured using the Bovine IFN-γ ELISA Development Kit (Mabtech, Nacka Strand, Sweden) and Bovine TNF-α ELISA Development Kit (Mabtech), respectively, according to the manufacturers’ protocols.

Techniques: Activation Assay, Isolation, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Nature Communications

Article Title: LANA-specific CD4 + effector T cells accumulate at the site of KSHV infection in humanized mice

doi: 10.1038/s41467-025-66992-2

Figure Lengend Snippet: A Schematic of primary TCR-transduced T cell generation for downstream assays. B IFNɣ levels of primary TCR-transduced T cells or normalized luminescence of TCR-transduced Jurkat Lucia NFAT cells co-cultured overnight with HLA-matched LCLs pulsed with decreasing peptide concentrations. Graph shows mean ± SD of n = 2 (Jurkat Cl33), n = 3 (Jurkat Cl156/12, primary Cl114) or n = 4 (rest) independent experiments. C IFNɣ ELISA of TCR-transduced T cells co-cultured with cognate peptide-pulsed or unpulsed HLA-matched LCLs. Boxplots represent 5 (Cl114, Cl33), 4 (Cl12) or 3 (Cl156) independent experiments, using T cells from 8 (Cl114, Cl33, Cl12) or 6 (Cl156) different donors, tested against 9 (Cl114), 6 (Cl33), 4 (Cl156), 3 (Cl12) different LCLs. D Frequency of CD107a + T cells after 6 h of co-culture with cognate peptide-pulsed or unpulsed HLA-matching LCLs. Boxplots represent 3 (Cl114, Cl33, Cl12) or 1 (Cl156) independent experiments, using T cells from 6 (Cl12), 4 (Cl114, Cl33) or 2 (Cl156) different donors, tested against 6 (Cl114), 5 (Cl33), 4 (Cl156) or 3 (Cl12) different LCLs. E Specific killing of HLA-matched pulsed or unpulsed LCLs after co-culture with TCR-transduced T cells. Boxplots represent 3 (Cl156, Cl33, Cl12) or 2 (Cl114) independent experiments, using T cells from 6 (Cl156, Cl33), 5 (Cl12) or 3 (Cl114) different donors, tested against 5 (Cl114, Cl156, Cl33) or 2 (Cl12) different LCLs. C–E Paired two-tailed t-test. Intracellular cytokine staining (6 h) and surface marker assessment (18 h) of CD4 + ( F ) and CD8 + ( G ) T cells after stimulation with PMA/Ionomycin, cognate peptide-pulsed or unpulsed LCLs or medium only as a negative control. Mean frequency of cells positive for the indicated marker (y-axis) in each condition (x-axis) is plotted from 3 (CD4 + ) or 1 (CD8 + ) independent experiments with 5 (CD4 + ) or 2 (CD8 + ) different T cell donors. Numerical means are shown for LCL-stimulated conditions. Asterisks indicate significance from paired two-sided t-tests comparing pulsed versus unpulsed LCLs. A–E * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Exact significant p ≥ 0.0001 from left to right (2D) 0.00015; (2E) p = 0.00236, p = 0.00019.

Article Snippet: IFNɣ or TNF was measured in the supernatant according to the manufacturer’s instructions (Human IFNɣ (HRP), Mabtech, 3420-1H; Human TNF (HRP), Mabtech, 3512-1H) and the signal was detected on an Infinite 200 PRO (Tecan).

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Co-Culture Assay, Two Tailed Test, Staining, Marker, Negative Control

Journal: Nature Communications

Article Title: LANA-specific CD4 + effector T cells accumulate at the site of KSHV infection in humanized mice

doi: 10.1038/s41467-025-66992-2

Figure Lengend Snippet: A Schematic of EBV&KSHV co-infected LCL (EK LCL) generation. B Flow cytometry histograms of GFP expression in different LCLs. C Western blot of whole-cell lysates from iSLK.219 and Brk.219 (± lytic induction) and from EBV-only or puromycin-selected EK LCLs (4 donors) stained for LANA and Tubulin. Tubulin-normalized LANA quantification indicated below blots. D IFNɣ production by TCR-transduced T cells co-cultured with HLA-matched LCLs ( ± KSHV infection), normalized to untransduced controls. Boxplots representing five (Cl33), four (Cl114, Cl12) or three (Cl156) independent experiments using T cells from eight (Cl114, Cl33, Cl12) or six (Cl156) donors, tested against six (Cl114, Cl33), four (Cl156) or three (Cl12) LCLs. Paired two-tailed t-test. E Frequencies of proliferating (CTV-diluted) or mouse CD19 + CD8 + T cells after seven-day co-culture of Cl12-transduced CD8 + T cells with irradiated HLA-matched or mismatched LCLs (± KSHV infection, ± peptide-pulsed), normalized to untransduced T cells. Boxplots representing two independent experiments with four T-cell and two target-cell donors. Linear mixed model fit by REML, T cell and LCL donor as a random effect, Holm-adjusted Tukey test. F IFNɣ secretion by T cells co-cultured with peptide-pulsed, KSHV-infected, or untreated MC116 B cells, or medium only. IFNɣ levels are shown relative to PMA/Ionomycin controls. Median ± SD of 2 independent experiments using T cells from three (Cl12) or four (rest) donors. Two-way ANOVA, Tukey’s multiple comparisons test, multiplicity adjusted p -values. G IFNɣ ELISA of TCR-transduced T cells co-cultured with autologous B cells infected with EBV or EBV&KSHV on days 1, 5, 14 post-infection. Mean of one experiment with two donor pairs. H IFNɣ ELISA of untransduced T cells incubated 72 h with conditioned media from freshly infected B cells. Mean ± SD of one experiment with three donor pairs. One-way ANOVA on log 10 -transformed data, Tukey’s multiple comparisons test. ( A–H ) * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Exact significant p ≥ 0.0001 (left to right, top to bottom): (3D) p = 0.03852; (3E) p = 0.00034; (3 F) Cl156: p = 0.0078, p = 0.0013, p = 0.0107; Cl33: p = 0.0224; Cl114: p = 0.0402, p = 0.0472; Cl12: p = 0.0016; (3H) p = 0.0037.

Article Snippet: IFNɣ or TNF was measured in the supernatant according to the manufacturer’s instructions (Human IFNɣ (HRP), Mabtech, 3420-1H; Human TNF (HRP), Mabtech, 3512-1H) and the signal was detected on an Infinite 200 PRO (Tecan).

Techniques: Infection, Flow Cytometry, Expressing, Western Blot, Staining, Cell Culture, Two Tailed Test, Co-Culture Assay, Irradiation, Enzyme-linked Immunosorbent Assay, Incubation, Transformation Assay

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

doi: 10.1007/978-1-4939-8567-8

Figure Lengend Snippet: Fig. 1 Initial Elispot Harmonization guidelines, as published in [4]

Article Snippet: Commercially ready to use human IL-4 ELISpot kit (Mabtech, Sweden).

Techniques: Enzyme-linked Immunospot

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

doi: 10.1007/978-1-4939-8567-8

Figure Lengend Snippet: Fig. 2 Steps of an Elispot experiment. The first step pertains to the preparation of the sample (guidance available in [6, 25]), which may include cell isolation (e.g., PBMC from whole blood), freezing, thawing, resting, counting and viability assessment, and perhaps isolation of subpopulations or expansion of cells. The second step, the assay (guidance available in [25, 31]), typically includes the coating and blocking of the Elispot plate, the plating of the sample and stimu- lants, incubation, and removal of cells and spot development. The third step, labeled by a star above the box, includes the enumeration of spots (guidance available in [24, 32]), and the analysis of raw spot numbers for obtaining mea- sures for response definition (guidance available in [33, 34]). This article focuses on the first part of step 3, the spot enumeration

Article Snippet: Commercially ready to use human IL-4 ELISpot kit (Mabtech, Sweden).

Techniques: Enzyme-linked Immunospot, Cell Isolation, Isolation, Blocking Assay, Incubation, Labeling

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

doi: 10.1007/978-1-4939-8567-8

Figure Lengend Snippet: Fig. 3 True and artificially altered well images. Images on the left hand side were taken with a Zeiss reader, with the RGB alignment and exposure settings optimally set as demonstrated in Fig. 4. The real specimen (Elispot plate) exposed high bluish-grey background staining, caused by typical overstaining with BCIP/NBT, and faint spots which were barely recognizable by eye. The upper panel image was taken from a well contain- ing cells stimulated with an antigen. The negative control (cells plus medium only) is shown in the lower panel. The right side images were obtained with a different reader system that performed automated color and intensity adjustments to artificially change the image to highly intense blue staining with nonrealistically strong spots, a process that was not controlled appropriately. Due to the falsely increased presentation of signals (and high sensitivity settings), counted spot numbers in the antigen-stimulated well and the negative control are inaccurately high. While perhaps looking more pleasing to the eye; the images on the right hand side are deceitful and do not reflect the true conditions of that Elispot plate. A part of the well periphery was also cut out and is not represented in the image

Article Snippet: Commercially ready to use human IL-4 ELISpot kit (Mabtech, Sweden).

Techniques: Enzyme-linked Immunospot, Staining, Negative Control

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

doi: 10.1007/978-1-4939-8567-8

Figure Lengend Snippet: Fig. 4 Optimal camera adaption of exposure and RGB alignment. The intensity of each of the three light chan- nels (RGB) is converted onto a grey scale that per definition has the lowest value set as zero (black, see arrow at left side of diagram) and the highest value at 255 (white, see arrow at right side of diagram). The camera should be adjusted so that all three signals have the same intensity (arrow pointing to the tops of the RGB curves). If this is not the case, the image may appear more red, blue, or green, depending on which signal has the highest intensity. Elispot reader systems have typically a command that allows the automated adjustment of the signal strength and the alignment of the RGB channels (also called Auto-White Balance = AWB). Further, the exposure determines how dark or bright an image will appear. It is important to adjust the exposure set- tings in a way that none of the signals from the RGB channels are lost in overexposure (“too bright,” arrow pointing to the right end bottom of RGB signal curves). Underexposure leads to too dark images

Article Snippet: Commercially ready to use human IL-4 ELISpot kit (Mabtech, Sweden).

Techniques: Enzyme-linked Immunospot

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

doi: 10.1007/978-1-4939-8567-8

Figure Lengend Snippet: Fig. 5 Concave well membrane. A well image from an old format MAHA well plate with many spots is shown. The image was taken with a KS Elispot reader system (Carl Zeiss, Inc., Thornwood, NY, USA). While spots in the well center are in focus, the well is shaped highly concave with the well periphery being completely out of focus and spots cannot be accurately discriminated and counted

Article Snippet: Commercially ready to use human IL-4 ELISpot kit (Mabtech, Sweden).

Techniques: Membrane, Enzyme-linked Immunospot

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

doi: 10.1007/978-1-4939-8567-8

Figure Lengend Snippet: Fig. 6 Elispot well with artifact ring. PBMC were tested for IFNɣ release. A ring of small artifacts is recognizable in the well periphery (arrow), indicative of cell death. A few true spots are also detectable, among a faint cover of artifacts across the membrane. It is advisable to minimize the AOI to exclude the artifact ring in the well periphery. The image was taken with a KS Elispot reader system (Carl Zeiss, Inc., Thornwood, NY, USA)

Article Snippet: Commercially ready to use human IL-4 ELISpot kit (Mabtech, Sweden).

Techniques: Enzyme-linked Immunospot, Membrane

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

doi: 10.1007/978-1-4939-8567-8

Figure Lengend Snippet: Fig. 8 Example of a B-cell Elispot well testing human B cells for IgG release. The well was coated with the antigen (whole protein) of interest. Spots are very large, requiring algorithm settings focusing on detecting large spots, combing small staining differences within a spot to one spot. The image was taken with an AID iSpot Reader system (Strassberg, Germany)

Article Snippet: Commercially ready to use human IL-4 ELISpot kit (Mabtech, Sweden).

Techniques: Enzyme-linked Immunospot, Staining

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

doi: 10.1007/978-1-4939-8567-8

Figure Lengend Snippet: Fig. 12 DNA precipitates. The well image contains a large netlike artifact that was caused by a DNA precipitate. Two blue spots can also be seen above the artifact. The image was taken with a KS Elispot reader system (Carl Zeiss, Inc., Thornwood, NY)

Article Snippet: Commercially ready to use human IL-4 ELISpot kit (Mabtech, Sweden).

Techniques: Enzyme-linked Immunospot

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

doi: 10.1007/978-1-4939-8567-8

Figure Lengend Snippet: Fig. 13 Granulocyte contamination. Two well images are presented (left image developed with TMB, right image developed with AEC) from PBMC testing. High levels of white ghost spots can be recognized in both wells, and spot disintegration is evident. It is very challenging to adjust parameters to obtain accurate spot counts. It is recommended to set the algorithm with focus on large spots, in order to allow the software to “repair” some of the spot disintegration and to avoid multiple counts for the same spot. The images were taken by a KS Elispot reader system (Carl Zeiss, Inc., Thornwood, NY)

Article Snippet: Commercially ready to use human IL-4 ELISpot kit (Mabtech, Sweden).

Techniques: Software, Enzyme-linked Immunospot

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

doi: 10.1007/978-1-4939-8567-8

Figure Lengend Snippet: Fig. 15 Changing amount of artifacts within a sample. Four well images present different stimulation condi- tions for the same PBMC sample in an IFNɣ Elispot. There are high amount of artifacts present in the negative control as well as in two different stimulations with peptide antigen. When cells are stimulated with SEB, the amount of artifacts decreases dramatically, and many well-defined spots are visible. The images were taken with a KS Elispot reader system (Carl Zeiss, Inc., Thornwood, NY)

Article Snippet: Commercially ready to use human IL-4 ELISpot kit (Mabtech, Sweden).

Techniques: Enzyme-linked Immunospot, Negative Control

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

doi: 10.1007/978-1-4939-8567-8

Figure Lengend Snippet: Fig. 1 Anatomy of a typical spot. After finishing the ELISpot assay developed with BCIP/NBT chromogen and immobilizing cells to the PVDF membrane, the mem- brane was removed from the plate and sectioned on the cryostat in a perpen- dicular direction. DAPI was used to counterstain the nuclei of immobilized cells (arrowhead). Spot boundaries indicate that cell-secreted proteins can migrate away from the cell in distances that exceed the size of the cell 3–5 times

Article Snippet: Commercially ready to use human IL-4 ELISpot kit (Mabtech, Sweden).

Techniques: Enzyme-linked Immunospot, Membrane

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

doi: 10.1007/978-1-4939-8567-8

Figure Lengend Snippet: Fig. 3 Example of nonspecific spots generated by cells sticking to PVDF membrane in the ELISpot plate. Cells sticking to the PVDF membrane in the ELISpot plate can be the source of a signal that resembles spots formed by cell-secreting proteins. This image illustrates nonspecific spots using HRP-AEC chromogenic detection (red color)

Article Snippet: Commercially ready to use human IL-4 ELISpot kit (Mabtech, Sweden).

Techniques: Generated, Membrane, Enzyme-linked Immunospot

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

doi: 10.1007/978-1-4939-8567-8

Figure Lengend Snippet: Fig. 5 Controls recommended for running side by side with experimental groups. Several controls are recom- mended to make sure that experimental conditions and reagents are not causing the formation of nonspecific spots: (1) Background control—complete ELISpot development with no cells added to wells; (2) No-detection antibody control—cells are added to the wells but ELISpot development omits adding detection antibodies; (3) Non-stimulated cells control—complete ELISpot development using non-stimulated cells (see Note 12); and (4) Immunoassay control—complete ELISpot development adding a corresponding recombinant protein rather than stimulated cells

Article Snippet: Commercially ready to use human IL-4 ELISpot kit (Mabtech, Sweden).

Techniques: Control, Enzyme-linked Immunospot, Recombinant

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

doi: 10.1007/978-1-4939-8567-8

Figure Lengend Snippet: Fig. 1 Effects of illumination conditions on the results of the automated search process. (a) Field of view in bottom of the ELISPOT well is evenly illuminated which results in almost a uniform background. (b) Illumination is uneven and as a result, the software easily detects sharp round spots, whereas blurry spots that have irregu- lar form may remain undetected

Article Snippet: Commercially ready to use human IL-4 ELISpot kit (Mabtech, Sweden).

Techniques: Enzyme-linked Immunospot, Software

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

doi: 10.1007/978-1-4939-8567-8

Figure Lengend Snippet: Fig. 2 Spots color subdivision for ELISPOT images. Left panel—the image with large dark blue spots and small red spots. Five blue and eight red spots were selected. Right panel—image with small brown and gray spots. Eight gray and 40 brown spots were selected

Article Snippet: Commercially ready to use human IL-4 ELISpot kit (Mabtech, Sweden).

Techniques: Enzyme-linked Immunospot

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

doi: 10.1007/978-1-4939-8567-8

Figure Lengend Snippet: Fig. 1 A schematic flowchart of ELISpot assay to detect Ag-specific ASCs. (A) Pre-coat the wells of the ELISpot plate with an Ag, e.g., tetanus toxin. (B) Seed serial diluted PBMCs into wells of the plate, respectively. Culture overnight (minimum: 8 h). (C) Wash off cells with PBS-T. (D) Add AP-conjugated detection Abs specific to IgM, IgG, or IgA. (E) Wash off unbound Abs. (F) Develop the spots with BCIP/NBT substrate solution

Article Snippet: Commercially ready to use human IL-4 ELISpot kit (Mabtech, Sweden).

Techniques: Enzyme-linked Immunospot

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

doi: 10.1007/978-1-4939-8567-8

Figure Lengend Snippet: Fig. 2 Representative ELISpot images of tetanus toxin-specific IgG ASCs detected from PBMCs. PBMCs were isolated from a healthy donor 2 weeks after receiving a booster of tetanus toxin. ELISpot plate was pre-coated with 50 μL/well of tetanus toxin (10 μg/mL in PBS). Aliquots of 5 × 105, 2.5 × 105, and 1.25 × 105 PBMCs (#1, #2 and #3) were seeded into wells of the ELISpot plate, respectively. Tetanus toxin-specific IgG ASCs were detected and illustrated

Article Snippet: Commercially ready to use human IL-4 ELISpot kit (Mabtech, Sweden).

Techniques: Enzyme-linked Immunospot, Isolation

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

doi: 10.1007/978-1-4939-8567-8

Figure Lengend Snippet: Fig. 1 Flowchart detailing the ELISPOT protocol at a glance

Article Snippet: Commercially ready to use human IL-4 ELISpot kit (Mabtech, Sweden).

Techniques: Enzyme-linked Immunospot

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

doi: 10.1007/978-1-4939-8567-8

Figure Lengend Snippet: Fig. 2 Examples of TNTC, positive, and negative ELISPOT wells. After development and drying, ELISPOT wells were imaged using an ELISPOT plate reader. The first well is an example of a well that is too numerous to count, or TNTC. The second well is a positive well with distinct and easily separated spots. The third well is a negative well containing only cell debris and no spots

Article Snippet: Commercially ready to use human IL-4 ELISpot kit (Mabtech, Sweden).

Techniques: Enzyme-linked Immunospot

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

doi: 10.1007/978-1-4939-8567-8

Figure Lengend Snippet: Fig. 1 A typical cultured ELISpot response to the panel of DENV serotype specific peptides. (a) The layout of the ELISpot for the 17 DENV serotype specific peptides is shown, with each peptide response done in duplicate. (b) A response to these peptides from one dengue seropositive donor is shown with responses to DENV2 pep- tide 11 and 18, DENV4 peptide 5 and 19 and PHA. Therefore, as this individual responds to SS peptides of DENV2 and DENV4, it is likely that he/she has been infected with these two DENV serotypes in the past

Article Snippet: Commercially ready to use human IL-4 ELISpot kit (Mabtech, Sweden).

Techniques: Cell Culture, Enzyme-linked Immunospot, Infection

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

doi: 10.1007/978-1-4939-8567-8

Figure Lengend Snippet: Fig. 1 A typical ex vivo ELISpot assay with PHA as the positive control, DENV NS3 overlapping peptides and media as the negative control

Article Snippet: Commercially ready to use human IL-4 ELISpot kit (Mabtech, Sweden).

Techniques: Ex Vivo, Enzyme-linked Immunospot, Positive Control, Negative Control

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

doi: 10.1007/978-1-4939-8567-8

Figure Lengend Snippet: Fig. 1 Typical IFN-γ ELISpot images showing antigen-specific responses to serotypes of DENV E protein from a representative DENV-immune and a DENV-naïve subject

Article Snippet: Commercially ready to use human IL-4 ELISpot kit (Mabtech, Sweden).

Techniques: Enzyme-linked Immunospot

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

doi: 10.1007/978-1-4939-8567-8

Figure Lengend Snippet: Fig. 2 IFN-γ responses measured by the dendritic cell-based ELISpot correlated with the immune status of the subjects suggesting the assay is antigen specific. SPUs to dengue serotype-specific E proteins in seven immune subjects (a) and seven naïve subjects (b)

Article Snippet: Commercially ready to use human IL-4 ELISpot kit (Mabtech, Sweden).

Techniques: Enzyme-linked Immunospot

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

doi: 10.1007/978-1-4939-8567-8

Figure Lengend Snippet: Fig. 1 Structure and sequences of the four multiple antigenic peptides (MAPs). The two MAPs on the left (top and bottom) consist of FIV p24 peptides (Fp9-3 and Fp14-3/4), and those on the right consist of FIV reverse transcriptase (RT) peptides (FRT3-3/4 and FRT7-1/2). The sequences of the long peptide and its smaller over- lapping peptides are shown for Fp14-3/4 (Fp14-3, Fp14-4), FRT3-3/4 (FRT3-3, FRT3-4), and FRT7-1/2 (FRT7- 1, FRT7-2). Four identical FIV peptides are on the amino-end of the MAP attached to a branched lysine backbone with palmitic acid (Pam) on the carboxyl-end. The final MAP vaccine consisted of all four MAPs at equal amounts (100 μg/dose each; total 400 μg/dose). Each MAP and its long and short peptides were used as FIV peptide stimulants in the IL2, IFNγ, and IL10 ELISpot assays and the T-cell proliferation analysis

Article Snippet: Commercially ready to use human IL-4 ELISpot kit (Mabtech, Sweden).

Techniques: Reverse Transcription, Enzyme-linked Immunospot

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

doi: 10.1007/978-1-4939-8567-8

Figure Lengend Snippet: Fig. 1 Influenza A/H1N1-specific memory B cell response pre- and post-influenza vaccination. Figure 1 demonstrates the dynamics of influenza-specific memory B cell ELISPOT response after influenza vaccination (in a cohort of 106 healthy older adults, 50–74 years old) and has been previously published in Viral Immunology [7] and Plos One [8]. The top (bottom) of the box indicates the 75th (25th) percentiles, respectively, while the bold line within the box indicates the median. The “whiskers” extend up to 1.5 times the interquartile range above or below the 75th or 25th percentiles, respectively. Beyond that point, individual points are plotted. B cell ELISPOT counts representing the influenza A/H1N1- specific IgG-producing memory-like B cell response plotted for each timepoint as spot forming units (SFUs) per 2 × 105 PBMCs

Article Snippet: Commercially ready to use human IL-4 ELISpot kit (Mabtech, Sweden).

Techniques: Enzyme-linked Immunospot

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

doi: 10.1007/978-1-4939-8567-8

Figure Lengend Snippet: Fig. 3 Example of programming: The figure represents a short impression how the automated script of the ELISpot assay can be programmed. Modules like “sub-routine” or “group” are included, as well as variables, loops, and the standard basic commands “aspirate” and “dispense”

Article Snippet: Commercially ready to use human IL-4 ELISpot kit (Mabtech, Sweden).

Techniques: Enzyme-linked Immunospot

Journal: Journal for Immunotherapy of Cancer

Article Title: Intraperitoneal CF33-hNIS combined with PD-L1 blockade eradicates gastric cancer peritoneal metastases and prevents recurrence via durable T cell memory

doi: 10.1136/jitc-2025-014530

Figure Lengend Snippet: Replication and cytotoxicity of CF33-hNIS in human and mouse gastric cancer (GC) cell lines and CD274/PD-L1 expression of mouse GC cell lines. (A, B) Seven GC cell lines, including five human GC cell lines (AGS, MKN74, KATO III, MKN45, and SUN-16 ( A )) and two mouse GC cell lines (ACKPY3944 and ACKPY4113 ( B )) were infected with CF33-hNIS (MOI=0.01) for 120 hours. Virus titers in the harvested cell lysates were determined using a standard plaque assay (n=3, each). (C, D) Five human GC cell lines ( C ) and two mouse GC cell lines ( D ) were infected with CF33-hNIS at MOIs 0.01, 0.1, 1, or 10. Cell survival relative to mock-infected cells was calculated daily postinfection for 8 days (n=3, each). (E, F) Two mouse GC cell lines ACKPY3944 ( E ) and ACKPY4113 ( F ) were treated with PBS, IFNβ1 (20 ng/mL) or IFNγ (20 ng/mL) for 18 hours. Cells were harvested and stained with PE-anti-mouse PD-L1 antibody or PE-isotype control antibody and analyzed by flow cytometry. The right statistical results show mean fluorescence intensity (MFI) of PD-L1 expression (n=3 or n=5). Data are shown as mean±SEM and analyzed using one-way or two-way ANOVA and Student’s t-test. ANOVA, analysis of variance; hNIS, human sodium iodide symporter; PBS, phosphate-buffered saline; MOI, multiplicity of infection; PFU, plaque-forming unit.

Article Snippet: Then, cells in the plate were removed by washing, and 100 μL of biotinylated anti-mouse IFNγ detection antibody (MABTECH, Catalog#3321-4HPT-2, Clone#R4-6A2) was added to each well for a 2-hour incubation at room temperature followed by subsequent washes.

Techniques: Expressing, Infection, Virus, Plaque Assay, Staining, Control, Flow Cytometry, Fluorescence, Saline

Journal: Journal for Immunotherapy of Cancer

Article Title: Intraperitoneal CF33-hNIS combined with PD-L1 blockade eradicates gastric cancer peritoneal metastases and prevents recurrence via durable T cell memory

doi: 10.1136/jitc-2025-014530

Figure Lengend Snippet: Memory T cell populations increase in peritoneal cavities following rechallenge in CTR mice. Mice that achieved CTR in Combo 2 treatment group (IP CF33-hNIS 1×10 8 + IP anti-PD-L1, n=6) were rechallenged with IP 1×10 6 ACKPY3944-ffluc cells on day 141 after initial tumor implantation (defined as day 0 post-rechallenge). (A) Experimental timeline showing tumor rechallenge and tissue collection timepoints. (B) Bioluminescence images showing tumor burden captured twice weekly. (C) Representative flow cytometry plots demonstrating memory T cell population changes in peritoneal lavage samples on days 11 and 30 post-rechallenge compared with healthy control (n=3, each group). (D) Quantification of total T cells and memory T cell subsets (n=3, each group). *p<0.05; **p<0.01 (E) IFNγ ELISpot assay measuring functional cell response in peritoneal cells harvested from healthy controls (n=3) or rechallenged mice (n=3, ear tag #978, #982, and #935) on day 30. Peritoneal cells were co-cultured with irradiated ACKPY3944 cells (E:T=16:1) in duplicate wells for 3 days. Data are shown as mean±SEM and analyzed using Student’s t-test. CTR, complete tumor regression; E:T, effector-to-target; hNIS, human sodium iodide symporter; IP, intraperitoneal; ROI, region of interest.

Article Snippet: Then, cells in the plate were removed by washing, and 100 μL of biotinylated anti-mouse IFNγ detection antibody (MABTECH, Catalog#3321-4HPT-2, Clone#R4-6A2) was added to each well for a 2-hour incubation at room temperature followed by subsequent washes.

Techniques: Tumor Implantation, Flow Cytometry, Control, Enzyme-linked Immunospot, Functional Assay, Cell Culture, Irradiation

Journal: Journal for Immunotherapy of Cancer

Article Title: Intraperitoneal CF33-hNIS combined with PD-L1 blockade eradicates gastric cancer peritoneal metastases and prevents recurrence via durable T cell memory

doi: 10.1136/jitc-2025-014530

Figure Lengend Snippet: Increased memory T cells in the spleen after in CTR mice. CTR mice in the Combo 2 treatment group (IP CF33-hNIS 1×10 8 + IP anti-PD-L1) were rechallenged with IP 1×10 6 ACKPY3944-ffluc cells on day 141 after initial tumor implantation (defined as day 0 post-rechallenge). Spleens were harvested from healthy control mice (n=3) and day 30 mice (n=3) post-rechallenge and analyzed for T cell subsets and functional responses. (A) Representative flow cytometry plots of memory T cells in spleen. (B) Percentages of total T cells and memory T cell subsets. *p<0.05; **p<0.01 (C) IFNγ ELISpot assay performed using splenocytes cocultured with irradiated ACKPY3944 cells (E:T=16:1) in duplicate wells for 3 days. Results are shown as number of IFNγ-producing cells per well. Data are shown as mean±SEM and analyzed using Student’s t-test. CTR, complete tumor regression; hNIS, human sodium iodide symporter; E:T, effector-to-target; IP, intraperitoneal; Tcm, central memory T cells; Tem, effector memory T cells; Tn, naïve T cells.

Article Snippet: Then, cells in the plate were removed by washing, and 100 μL of biotinylated anti-mouse IFNγ detection antibody (MABTECH, Catalog#3321-4HPT-2, Clone#R4-6A2) was added to each well for a 2-hour incubation at room temperature followed by subsequent washes.

Techniques: Tumor Implantation, Control, Functional Assay, Flow Cytometry, Enzyme-linked Immunospot, Irradiation

Journal: NPJ Vaccines

Article Title: Strong and early immune responses against SARS-CoV-2 in mice and rhesus macaques after BNT162b3 vaccination

doi: 10.1038/s41541-025-01365-w

Figure Lengend Snippet: Female BALB/c mice ( n = 5–8) received a single i.m. dose of 1 µg BNT162b1 or 0.2 µg or 1 µg BNT162b3 or buffer control. Serum samples obtained after vaccination for a follow-up period of 28 days were analyzed for a concentration of RBD-binding IgG, and for b pVNT 50 (log 10 ) against the wild-type SARS-CoV-2 lineage. Horizontal gray line marks the LLOD. c IFN-γ secretion by splenocytes from Day 28 pulsed with RBD overlapping peptide pool, by ELISpot. d Secretion of cytokines by splenocytes from Day 28 pulsed with S1 overlapping peptide pool, determined on culture supernatants by bead-based multiplex analysis. P values, α = 0.05, from two- ( a , b ) or one-way ANOVA followed by Tukey’s multiple comparisons test ( c [BNT162b3], d ), or unpaired t -test ( c , BNT162b1). Symbols represent individual animals; bar heights indicate group arithmetic means ( c , d ) or geometric means ( a , b written above bars). ELISpot enzyme-linked immunosorbent spot, GM-CSF granulocyte-macrophage colony-stimulating factor, IFN-γ interferon gamma, IgG immunoglobulin G, IL interleukin, i.m. intramuscular, pVNT 50 pseudovirus-based VSV-SARS-CoV-2 50% neutralization titers, RBD receptor-binding domain, S spike, S1 N-terminal furin cleavage fragment of S, SARS-CoV-2 severe acute respiratory syndrome coronavirus 2, T H T helper, TNF tumor necrosis factor, VSV vesicular stomatitis virus.

Article Snippet: Macaque PBMCs were tested with commercially available NHP IFN-γ and IL-4 ELISpot assay kits (Mabtech, cat. n. 3410-4HPW-10).

Techniques: Control, Concentration Assay, Binding Assay, Enzyme-linked Immunospot, Multiplex Assay, ELISpot Assay, Neutralization, Virus

Journal: NPJ Vaccines

Article Title: Strong and early immune responses against SARS-CoV-2 in mice and rhesus macaques after BNT162b3 vaccination

doi: 10.1038/s41541-025-01365-w

Figure Lengend Snippet: Female BALB/c mice ( n = 12) received two i.m. doses (Days 0 and 92) of 0.2 µg or 5 µg of BNT162b3 or 5 µg control RNA-LNP (C). Serum samples obtained for a follow-up period of 127 days were analyzed for a serum concentration of RBD-binding IgG, and for b pVNT 50 (log 10 ) against the wild-type SARS-CoV-2 lineage. Horizontal gray lines mark the LLODs. c IFN-γ secretion by splenocytes from Day 127 (Day 35 PD2) pulsed with RBD overlapping peptide pool, by ELISpot. P values, α = 0.05, from one-way ANOVA followed by Tukey’s multiple comparisons test ( c ). Symbols represent individual animals; bar heights indicate geometric ( a , b written above bars) or arithmetic means ( c ). ELISpot enzyme-linked immunosorbent spot, IFN-γ interferon gamma, i.m. intramuscular, LLOD lower limit of detection, RNA-LNP ribonucleic acid lipid nanoparticle, PD post dose, pVNT 50 pseudovirus-based VSV-SARS-CoV-2 50% neutralization titers, RBD receptor-binding domain, S spike, SARS-CoV-2 severe acute respiratory syndrome coronavirus 2, VSV vesicular stomatitis virus.

Article Snippet: Macaque PBMCs were tested with commercially available NHP IFN-γ and IL-4 ELISpot assay kits (Mabtech, cat. n. 3410-4HPW-10).

Techniques: Control, Concentration Assay, Binding Assay, Enzyme-linked Immunospot, ELISpot Assay, Neutralization, Virus

Journal: NPJ Vaccines

Article Title: Strong and early immune responses against SARS-CoV-2 in mice and rhesus macaques after BNT162b3 vaccination

doi: 10.1038/s41541-025-01365-w

Figure Lengend Snippet: Male macaques (2–4 years old, n = 6) received two i.m. doses of 30 μg of BNT162b1 or BNT162b3 (Day 0 and 21; arrowhead below the x -axes). a RBD-binding IgG concentration (LLOD = 1.1505 U mL −1 ). b VNT 50 (LLOD = 20) against the wild-type SARS-CoV-2 lineage. The blue triangles in a and b mark the mean values for previously published BNT162b1 data . Horizontal gray lines mark the LLOD. Values below the LLOD were set to 1/2 the LLOD. c–f PBMCs collected on Days 0, 14, and 28 or 42 after Dose 1 were pulsed ex vivo with a full-length S overlapping peptide pool. c , IFN-γ and IL-4 ELISpot. d Frequency of S-specific CD4 + T cells expressing IFN-γ, IL-4, IL-21, any T H 1 cytokine (IFN-γ, IL-2 or TNF-α) and all three T H 1 cytokines by flow cytometry. e Frequency of S-specific CD4 + T cells and cTfh cells expressing IL-21, and any T H 1 cytokine (IFN-γ, IL-2 or TNF-α) by flow cytometry. f Frequency of S-specific CD8 + T cells expressing IFN-γ by flow cytometry. Each symbol represents one macaque. Heights of bars indicate the geometric ( a , b ) or arithmetic ( c – f ) means for each group, and values are written above the bars. cTfh circulating T follicular helper, IFN-γ interferon gamma, IL interleukin, i.m. intramuscular, LLOD lower limit of detection, NT not tested, NA not available, PBMC peripheral blood mononuclear cell, RBD receptor-binding domain, RNA-LNP ribonucleic acid lipid nanoparticle, S spike, SARS-CoV-2 severe acute respiratory syndrome coronavirus 2, T H T helper, TNF-α tumor necrosis factor alpha, VNT 50 SARS-CoV-2 virus 50% neutralization titers.

Article Snippet: Macaque PBMCs were tested with commercially available NHP IFN-γ and IL-4 ELISpot assay kits (Mabtech, cat. n. 3410-4HPW-10).

Techniques: Binding Assay, Concentration Assay, Ex Vivo, Enzyme-linked Immunospot, Expressing, Flow Cytometry, Virus, Neutralization