Journal: International Journal of Molecular Sciences

Article Title: Quantifying PD1 Saturation by PDL1 in Tumor Tissue Using a Novel RNA Aptamer-Based Assay

doi: 10.3390/ijms27125469

Figure Lengend Snippet: Correlation between PD1 LIRECAP assay (C:P aptamer ratio) and PD1 signaling in the Jurkat-Raji co-culture system ( A ) Schematic illustration of the PD1 LIRECAP assay. Equimolar concentrations of P and C aptamers were added to biospecimens, bound aptamers extracted and aptamers amplified together using a single set of primers and quantified using unique TaqMan colorimetric probes. The ratio of bound aptamers (C:P ratio) was determined. ( B ) Raji-PDL1 and Raji-Null cells were combined in varying ratios (0–100% PDL1 positivity) and co-cultured with Jurkat-PD1 cells at a 1:1 Raji:Jurkat ratio. Jurkat-PD1 T cell inhibition mediated through the PD1 signaling was quantified as the percentage decrease in luciferase activity relative to the 0% Raji-PDL1 control in co-culture conditioned medium. After supernatant was collected, cell pellets were formalin-fixed and analyzed using the PD1 LIRECAP assay to assess the C:P ratio. ( C ) Raji-PDL1 cells correlate with inhibition of Jurkat-PD1 T cell activation (mean ± SEM from n = 4 independent sets). ( D ) Increasing the percentage of Raji-PDL1 increases the C:P ratio as determined by the PD1 LIRECAP assay (mean ± SEM from n = 4 independent sets). ( E ) C:P ratio as determined by the PD1 LIRECAP assay correlates with PD1 signaling-mediated T cell inhibition in the Raji-Jurkat co-culture system (mean ± SEM from n = 4 independent sets).

Article Snippet: The ratio of bound C18 to P17 (C:P ratio), as determined by the PD1 LIRECAP assay (schema in A), was evaluated using the Jurkat-Lucia TCR-hPD1 cell and Raji cell co-culture system (Invivogen, San Diego, CA, USA), which also enables evaluation of PD1 signaling.

Techniques: Co-culture Assay, Amplification, Cell Culture, Inhibition, Luciferase, Activity Assay, Control, Activation Assay, ATP Bioluminescent Assay, TaqMan Probe Assay

Journal: International Journal of Molecular Sciences

Article Title: Quantifying PD1 Saturation by PDL1 in Tumor Tissue Using a Novel RNA Aptamer-Based Assay

doi: 10.3390/ijms27125469

Figure Lengend Snippet: Correlation between PD1 LIRECAP assay (C:P aptamer ratio) and PD1–PDL1 proximity in FFPE biospecimens from Jurkat-Raji co-culture system ( A ) Schematic illustration of the preparation and analysis of FFPE sections from the Jurkat-Raji co-culture system used to assess the correlation between the PD1 LIRECAP assay and PD1–PDL1 proximity. ( B ) C:P ratio as measured by PD1 LIRECAP assay correlates with PD1 saturation by PDL1 (mean ± SEM from n = 2 independent sets) ( C ) Representative images from PD1–PDL1 PLA of FFPE from cell pellets (Blue—DAPI; Red—PLA positivity) ( n = 2). ( D ) Quantification of PLA positivity per cell, expressed as percent positive cells (mean ± SEM from n = 2 independent sets). ( E ) PD1–PDL1 PLA correlates with C:P aptamer ratio, as measured by PD1 LIRECAP assay (mean ± SEM from n = 2 independent sets).

Article Snippet: The ratio of bound C18 to P17 (C:P ratio), as determined by the PD1 LIRECAP assay (schema in A), was evaluated using the Jurkat-Lucia TCR-hPD1 cell and Raji cell co-culture system (Invivogen, San Diego, CA, USA), which also enables evaluation of PD1 signaling.

Techniques: Co-culture Assay, Analysis, Formalin-fixed Paraffin-Embedded, Proximity Ligation Assay

Journal: International Journal of Molecular Sciences

Article Title: Quantifying PD1 Saturation by PDL1 in Tumor Tissue Using a Novel RNA Aptamer-Based Assay

doi: 10.3390/ijms27125469

Figure Lengend Snippet: Selection of lead aptamer candidates for PD1 LIRECAP assay. ( A ) Schematic illustration of the SELEX approach used to generate enriched aptamer pools recognizing unoccupied PD1 (PD1 SELEX) and PD1–PDL1 complex (Complex SELEX). ( B ) Binding specificity of P aptamer pools to recombinant PD1, PDL1 and PD1–PDL1 complex. ( C ) Binding specificity of enriched C aptamer pools to recombinant PD1, PDL1 and PD1–PDL1 complex. Data shown are mean ± SEM of three independent experiments. ( D ) Percent enrichment of unique aptamer sequences in P and C aptamer pools following various rounds of selection as determined by Illumina-based high-throughput sequencing. ( E ) Fold enrichment of lead P aptamer candidates (P10, P17 and P150) based on copy number in the final enriched pool normalized to the starting library (Rd 0). ( F ) Fold enrichment of lead C aptamer candidates (C18, C52 and C113) based on copy number in the final enriched pool, normalized to the starting library (Rd 0).

Article Snippet: The Jurkat-Raji PD1/PDL1 assay (Bio-IC) co-culture system used for studying PD1–PDL1 interaction was purchased from Invivogen, Pittsburgh, PA, USA.

Techniques: Selection, Binding Assay, Recombinant, Next-Generation Sequencing

Journal: International Journal of Molecular Sciences

Article Title: Quantifying PD1 Saturation by PDL1 in Tumor Tissue Using a Novel RNA Aptamer-Based Assay

doi: 10.3390/ijms27125469

Figure Lengend Snippet: Specificity of lead P and C aptamer candidates toward PD1, PDL1 and PD1–PDL1 complex. Lead P aptamer candidates. ( A ) P10, ( B ) P17, and ( C ) P150 binding to unoccupied PD1, PDL1 and PD1–PDL1 complex. Lead C aptamer candidates ( D ) C18, ( E ) C52, and ( F ) C113 binding to PD1–PDL1 complex, unoccupied PD1 and PDL1. Data shown are mean ± SEM of three independent experiments.

Article Snippet: The Jurkat-Raji PD1/PDL1 assay (Bio-IC) co-culture system used for studying PD1–PDL1 interaction was purchased from Invivogen, Pittsburgh, PA, USA.

Techniques: Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: Quantifying PD1 Saturation by PDL1 in Tumor Tissue Using a Novel RNA Aptamer-Based Assay

doi: 10.3390/ijms27125469

Figure Lengend Snippet: Correlation between PD1 LIRECAP assay (C:P aptamer ratio) and PD1 signaling in the Jurkat-Raji co-culture system ( A ) Schematic illustration of the PD1 LIRECAP assay. Equimolar concentrations of P and C aptamers were added to biospecimens, bound aptamers extracted and aptamers amplified together using a single set of primers and quantified using unique TaqMan colorimetric probes. The ratio of bound aptamers (C:P ratio) was determined. ( B ) Raji-PDL1 and Raji-Null cells were combined in varying ratios (0–100% PDL1 positivity) and co-cultured with Jurkat-PD1 cells at a 1:1 Raji:Jurkat ratio. Jurkat-PD1 T cell inhibition mediated through the PD1 signaling was quantified as the percentage decrease in luciferase activity relative to the 0% Raji-PDL1 control in co-culture conditioned medium. After supernatant was collected, cell pellets were formalin-fixed and analyzed using the PD1 LIRECAP assay to assess the C:P ratio. ( C ) Raji-PDL1 cells correlate with inhibition of Jurkat-PD1 T cell activation (mean ± SEM from n = 4 independent sets). ( D ) Increasing the percentage of Raji-PDL1 increases the C:P ratio as determined by the PD1 LIRECAP assay (mean ± SEM from n = 4 independent sets). ( E ) C:P ratio as determined by the PD1 LIRECAP assay correlates with PD1 signaling-mediated T cell inhibition in the Raji-Jurkat co-culture system (mean ± SEM from n = 4 independent sets).

Article Snippet: The Jurkat-Raji PD1/PDL1 assay (Bio-IC) co-culture system used for studying PD1–PDL1 interaction was purchased from Invivogen, Pittsburgh, PA, USA.

Techniques: Co-culture Assay, Amplification, Cell Culture, Inhibition, Luciferase, Activity Assay, Control, Activation Assay, ATP Bioluminescent Assay, TaqMan Probe Assay

Journal: International Journal of Molecular Sciences

Article Title: Quantifying PD1 Saturation by PDL1 in Tumor Tissue Using a Novel RNA Aptamer-Based Assay

doi: 10.3390/ijms27125469

Figure Lengend Snippet: Correlation between PD1 LIRECAP assay (C:P aptamer ratio) and PD1–PDL1 proximity in FFPE biospecimens from Jurkat-Raji co-culture system ( A ) Schematic illustration of the preparation and analysis of FFPE sections from the Jurkat-Raji co-culture system used to assess the correlation between the PD1 LIRECAP assay and PD1–PDL1 proximity. ( B ) C:P ratio as measured by PD1 LIRECAP assay correlates with PD1 saturation by PDL1 (mean ± SEM from n = 2 independent sets) ( C ) Representative images from PD1–PDL1 PLA of FFPE from cell pellets (Blue—DAPI; Red—PLA positivity) ( n = 2). ( D ) Quantification of PLA positivity per cell, expressed as percent positive cells (mean ± SEM from n = 2 independent sets). ( E ) PD1–PDL1 PLA correlates with C:P aptamer ratio, as measured by PD1 LIRECAP assay (mean ± SEM from n = 2 independent sets).

Article Snippet: The Jurkat-Raji PD1/PDL1 assay (Bio-IC) co-culture system used for studying PD1–PDL1 interaction was purchased from Invivogen, Pittsburgh, PA, USA.

Techniques: Co-culture Assay, Analysis, Formalin-fixed Paraffin-Embedded, Proximity Ligation Assay

Journal: bioRxiv

Article Title: Cryoelectron tomography of HIV-1 cell-cell transmission conjugates reveals a secluded environment for viral assembly and transfer

doi: 10.64898/2026.06.11.725753

Figure Lengend Snippet: ( A ) Comparison NL4-3 Envelope and Δ Env sequences. Dotted box indicates sequence deletion and hashed box with red cross indicates lack of translation due to frameshift and introduction of a stop codon ( B ) The percentage of all (single-cell and conjugate) GFP+CTDR+ events at 3h or 24h co-cultures of uninfected SupT1 R5 cells mixed with either NL4-3 IRES-eGFP or Δ Env IRES-eGFP infected SupT1 R5 cells. ( C ) Representative imaging cytometry of single cells and conjugates present in NL4-3 IRES-eGFP and Δ Env IRES-eGFP co-cultures at 3h and 24h. ( D ) Breakdown of the distribution of single cell and conjugate GFP+CTDR+ events within the 3-hour or 24-hour co-cultures. Data from N=1 experiment that was performed in duplicate.

Article Snippet: After 3 hours, co-cultures were fixed with 4% PFA for 30 minutes at room temperature and permeabilized with saponin 0.1% (ThermoFisher, #J63209.AK) for 10 minutes, followed by a blocking step with 2% normal goat serum and 1% BSA for 15 minutes.

Techniques: Comparison, Sequencing, Single Cell, Infection, Imaging, Cytometry

Journal: bioRxiv

Article Title: Cryoelectron tomography of HIV-1 cell-cell transmission conjugates reveals a secluded environment for viral assembly and transfer

doi: 10.64898/2026.06.11.725753

Figure Lengend Snippet: ( A ) Representative dot plots of multicell conjugates in 3h co-cultures between target CTDR+ SupT1 R5 cells mixed with either NL4-3 IRES-eGFP or ΔEnv IRES-eGFP infected SupT1 R5 cells. ( B ) The percentage of GFP+CTDR+ multicell conjugate events from initial 0-hour or 3-hour co-cultures. ( C ) Representative dot plots of single cells in 24-hour co-cultures of target CTDR+ SupT1 R5 cells mixed with either NL4-3 IRES-eGFP or ΔEnv IRES-eGFP+ infected SupT1 R5 cells ( D ) The percentage of GFP+CTDR+ single cell events from initial 0-hour or 24-hour co-cultures. Distinct shapes in B and D each represent distinct biological replicates (N = 3) and each individual shape a technical replicate. (E-H) Same as A-D but with co-culture of infected GFP+ primary CD4+ T lymphocytes and target CTDR+ primary CD4+ T lymphocytes. Shapes in F and H represent distinct primary cell donors (N = 4). All values are normalized to initial GFP+ infection levels prior to co-culture. Values below x-axis indicate the mean and standard deviation for each category. Black bar represents the mean and the gray box represents ±1 standard deviation. P-values calculated using either two-tailed unpaired t- test ( B and D ) or two-tailed paired t- test ( F and H ).

Article Snippet: After 3 hours, co-cultures were fixed with 4% PFA for 30 minutes at room temperature and permeabilized with saponin 0.1% (ThermoFisher, #J63209.AK) for 10 minutes, followed by a blocking step with 2% normal goat serum and 1% BSA for 15 minutes.

Techniques: Infection, Single Cell, Co-Culture Assay, Standard Deviation, Two Tailed Test

Journal: bioRxiv

Article Title: Cryoelectron tomography of HIV-1 cell-cell transmission conjugates reveals a secluded environment for viral assembly and transfer

doi: 10.64898/2026.06.11.725753

Figure Lengend Snippet: ( A , D ) Quantification of GFP+CTDR+ multicell conjugates events in 3-hour co-cultures of target CTDR+ SupT1 R5 and NL4-3 IRES-eGFP infected SupT1 R5 cells treated with varying concentrations of soluble CD4 (sCD4, A ) or anti-CD4 (SIM.2, D ). ( B and E ) Quantification of GFP+CTDR+ single cells in 24-hour co-cultures treated with varying concentrations of sCD4 ( B ) or SIM.2 ( E ). ( C and F ) Quantification of GFP+ SupT1 R5 after cell-free NL4-3 IRES-eGFP virus infection treated with varying concentrations of sCD4 ( C ) or SIM.2 ( F ). Data are derived from distinct biological replicates (N = 2), where colors represent distinct biological and each dot a technical replicate. All values are normalized to untreated controls and initial GFP+ infection levels prior to co-culture (if applicable). Values below x-axis indicate the mean and standard deviation for each category. Black bar represents the mean and the gray box represents ±1 standard deviation. P-values calculated using one-way ANOVA with a Tukey’s test for multiple comparisons.

Article Snippet: After 3 hours, co-cultures were fixed with 4% PFA for 30 minutes at room temperature and permeabilized with saponin 0.1% (ThermoFisher, #J63209.AK) for 10 minutes, followed by a blocking step with 2% normal goat serum and 1% BSA for 15 minutes.

Techniques: Infection, Virus, Derivative Assay, Co-Culture Assay, Standard Deviation

Journal: Advanced Science

Article Title: PANX2 Suppresses Lung Adenocarcinoma Progression by Inducing Disulfidptosis and Enhancing Antitumor Immunity

doi: 10.1002/advs.75662

Figure Lengend Snippet: PANX2 overexpression in LUAD cells activates antitumor immunity via eATP‐P2X7R signaling. (A) ELISA of cytokines in co‐culture supernatants from PANX2‐OE/KO co‐cultures vs. respective controls. (B) Flow cytometry immunophenotyping: percentage of CD8 + T cells in total T cells from PANX2‐OE/KO co‐cultures vs. controls. (C) Flow cytometry: percentage of CD107a + cytotoxic subset in CD8 + T cells from PANX2‐OE/KO co‐cultures vs. controls. (D) Chemiluminescence assay of extracellular ATP release in PANX2‐OE/KO A549 cells vs. controls. (E) Chemiluminescence assay of ATP concentration modulation by PANX2‐OE/KO in A549 cells within the co‐culture microenvironment vs. controls. (F) WB to assess P2X7R protein levels in hPBMCs co‐cultured with PANX2‐OE/KO A549 cells vs. controls. (G) ATP assay to evaluate the reversal effect of Apyrase (10 U/mL) or A438079 (10 µM) on PANX2‐OE‐increased ATP levels in co‐cultures after 72 h (heat‐inactivated Apyrase as control). (H) WB: Apyrase (10 U/mL) or A438079 (10 µM) reverses PANX2‐OE‐increased P2X7R protein levels in hPBMCs after 72 h co‐culture (heat‐inactivated (HI)‐Apyrase as control). (I) ELISA: Apyrase (10 U/mL) or A438079 (10 µM) reverses PANX2‐OE‐increased cytokine secretion in co‐cultures (HI‐Apyrase as control). (Data are presented as means ± SD; n ≥ 3 independent experiments; “OE,” “KO,” and “CON” denote overexpression, knockout, and control, respectively).

Article Snippet: To inhibit the ATP–P2X7R signaling, co‐cultures were treated with the P2X7R antagonist A438079 (10 μM; AbMole, M11483) or the ATP‐hydrolyzing enzyme Apyrase (10 U/mL; MCE, HY‐P2764) for 72 h. A control group received heat‐inactivated Apyrase (95°C for 15 min) under identical conditions.

Techniques: Over Expression, Enzyme-linked Immunosorbent Assay, Co-Culture Assay, Flow Cytometry, Chemiluminescence Immunoassay, Concentration Assay, Cell Culture, ATP Assay, Control, Knock-Out