aid classic elispot plate reader  (Cellular Technology Ltd)

Journal: iScience

Article Title: Extensive evolution and T cell escape by SARS-CoV-2 in a 2.5-year persistent infection of an immunocompromised host

doi: 10.1016/j.isci.2026.114917

Figure Lengend Snippet: Magnitude and specificity of T cell memory responses generated by 899-day infection (A) Ex vivo IFNγ-ELISpot well images. DMSO control unstimulated wells and wells stimulated with overlapping peptides covering S1 and S2 region of Spike (duplicates shown), or single peptides S51 and S134. (B) Total magnitude of SARS-CoV-2-specific memory T cell response to structural proteins Spike, membrane (M), nucleoprotein (NP) and open reading frame 3a (ORF3a) and RTC proteins (NSP7, NSP12 polymerase, and NSP13 helicase) colored by protein targeted and measured after persistent infection (>900 days). (C and D) Total magnitude of SARS-CoV-2-specific T cell response (C) or T cell response to Structural and RTC proteins (D) in pre-pandemic samples (pre-August 2019), in exposed healthcare workers (HCW) who remained seronegative, including abortive infections, and in HCW with laboratory confirmed SARS-CoV-2 infections (samples 4 months post-exposure/infection in June to July 2020) for comparison to T cell response in persistently infected patient. (E) Ratio of the magnitude of the T cell response to RTC/structural T cells. Percentage of cohort with a response above 1 (stronger response to RTC than structural proteins) shown below. (F) Magnitude of T cell response to a pool of epitopes from Flu, EBV, and CMV. (B–E) Subset of data previously published in Swadling et al. (A–F) IFNγ-ELISpot. (C and D) Box and Whisker, Tukey. (C–F) Statistical analysis was performed using Kruskal-Wallis tests with Dunn’s correction. ∗ p ≤ 0.05; ∗∗ p ≤ 0.01; ∗∗∗ p ≤ 0.001; ∗∗∗∗ p < 0.0001. (E and F) Bars, geomean.

Article Snippet: Plates were washed in double-distilled H 2 O and left to dry overnight before being read on the AID classic ELISpot plate reader (Immunospot S6 Universal M2 ELISpot Reader).

Techniques: Generated, Infection, Ex Vivo, Enzyme-linked Immunospot, Control, Membrane, Comparison, Whisker Assay

Journal: iScience

Article Title: Extensive evolution and T cell escape by SARS-CoV-2 in a 2.5-year persistent infection of an immunocompromised host

doi: 10.1016/j.isci.2026.114917

Figure Lengend Snippet: Failure of host T cells to recognize emerging virus (A) Ex vivo magnitude of the T cell response to individual ancestral sequence peptides in which mutation arose over persistent infection by IFNγ-ELISpot. (B) Magnitude of the CD4 and CD8 T cell responses after 10-day in vitro peptide expansion with individual ancestral sequence peptides. Percentage of CD4 and CD8 producing IFNγ, TNF, or both are shown. (C) Magnitude of IFNγ+, IFNγ+TNF+, and CTV lo IFNγ+ CD4 and CD8 T cells after 8-day expansion with a pool of 12 peptides corresponding to ancestral sequence epitopes or variant sequence epitopes containing mutations in the virus isolated at day 899 of infection. (D) Magnitude of the IFNγ+TNF+ CD4 or CD8 T cell response after 10-day in vitro peptide stimulation with individual epitopes using ancestral sequence or variant sequence peptides for culture and re-stimulation on day 9. Summary data showing all T cell responses that were detectable (left) or shown for individual peptides (right) for CD4 then CD8 T cells. Duplicate and triplicate stimulations are shown for CD8 T cells for M14 and NSP2, respectively. (D) Bars, mean. Statistical analysis was performed using Kruskal-Wallis tests with Dunn’s correction. ∗ p ≤ 0.05; ∗∗∗ p ≤ 0.001. Peptide sequences shown in .

Article Snippet: Plates were washed in double-distilled H 2 O and left to dry overnight before being read on the AID classic ELISpot plate reader (Immunospot S6 Universal M2 ELISpot Reader).

Techniques: Virus, Ex Vivo, Sequencing, Mutagenesis, Infection, Enzyme-linked Immunospot, In Vitro, Variant Assay, Isolation

Journal: Scientific Reports

Article Title: IgM + IgD − B cells in human gut-associated lymphoid tissue have memory features and give rise to IgM + and IgA + antibody-secreting cells

doi: 10.1038/s41598-025-11209-1

Figure Lengend Snippet: Spontaneous IgM secretion by tonsil and appendix B cells. ( A ) Representative images of ELISpot wells for spontaneous IgM secretion by tonsil and appendix IgM + B cell subpopulations after 6 h of incubation. ( B ) Quantification of ELISpot data from tonsil (left) and appendix (right). Each dot represents a single subject. Results are presented as mean ± SEM; two-tailed unpaired Student’s t-test, * P < 0.05. Gating strategy for cell sorting (Supplementary Fig. 5). Population purity > 97%.

Article Snippet: Spots were developed using the HRP substrate - TMB (MABTECH- Cat: 3651-10) and counted using an S6 FluoroCore M2 ELISpot plate reader (ImmunoSpot, CTL).

Techniques: Enzyme-linked Immunospot, Incubation, Two Tailed Test, FACS

Journal: iScience

Article Title: Hypoimmune CD19 CAR T cells treat allogeneic mice with features of spontaneous systemic lupus erythematosus

doi: 10.1016/j.isci.2025.112806

Figure Lengend Snippet: HIP CAR T cells do not induce an allogeneic immune response in NZB/W lupus mice (A) One million WT or HIP CAR T cells were intravenously injected into fully allogeneic NZB/W lupus mice and the host immune response was quantified after 6 days. CD8 T cells were recovered from the spleen of these mice, NK cells and macrophages were taken from C57BL/6 mice, and IgM and IgG donor CAR T cell-specific antibodies (DSA) were quantified. (B) Interferon-γ (IFN-γ) ELISpot assays were performed with CD8 lymphocytes isolated from the NZB/W recipients and the injected WT or HIP CAR T cells as stimulators. Spot frequencies were automatically enumerated (5 animals per group, all single animals are shown, mean ± SD, Mann-Whitney test). (C) Impedance cytotoxicity assays with WT and or HIP CAR T cells as targets and isolated CD8 lymphocytes from the lupus mice as effector cells (5 animals per group, mean ± SD per time point). The cell index is normalized at time point zero hours and a drop of the curve indicates target cell killing, while a stable signal indicates target cell survival. Fluctuations in the first few hours reflect cell culture perturbations from the addition of the effector cells. (D) IgM and IgG DSAs were quantified by flow cytometry (5 animals per group, all single animals are shown, mean ± SD, Mann-Whitney test). The background of this assay is shown in a dashed line. (E and F) Impedance cytotoxicity assays with WT or HIP CAR T cells or MHC class I and II-deficient double knockout (DKO) cells as targets and C57BL/6 NK cells (E) or macrophages (Mac; F) as effector cells (5 animals per group, mean ± SD per time point).

Article Snippet: One hundred thousand stimulator cells were incubated with one million recipient responder splenocytes for 24 h and IFN-γ spot frequencies were enumerated using an Elispot plate reader (AID Diagnostika GmbH, Strassburg, Germany).

Techniques: Injection, Enzyme-linked Immunospot, Isolation, MANN-WHITNEY, Cell Culture, Flow Cytometry, Double Knockout