cd8 t cell isolation kit  (Miltenyi Biotec)

Journal: bioRxiv

Article Title: Enhancing Patient Lymphocyte Response to Peritoneal Malignancies Using a Personalized Immunocompetent Microfluidic Co-Culture Platform

doi: 10.64898/2026.02.19.706377

Figure Lengend Snippet: (A) Comparison of the percent of polyfunctional cells (multiple cytokine secretions per cell) in total CD8 + cell populations between immune cell groups (PBMCe), (OIL), and (TIL)] for 4 patients (n=3 appendiceal, n=1 mesothelioma) measured by Isoplexis Single Cell Secretome. The mean percent of total polyfunctional cells across all patients is also depicted. (B) Single cell polyfunctional strength index scores (PSI; number of single cells producing the cytokine multiplied by the cytokine signal strength) from the same patients in A to characterize the predominate cytokines produced. Cytokines are grouped into effector (green: Granzyme B, IFN-γ, MIP-1α, Perforin, TNF-α), stimulatory (blue: GM-CSF, IL-2, IL-5, IL-8), chemoattractive (purple: MIP-1β), and regulatory functions (yellow: IL-4, TGF-β1). Box plots of both total PSI scores and PSI scores for each cytokine group are also shown for the combined patient groups. (C) Correlation heatmap for cytokine expression in the cytotoxicity assay co-culture media [relative fluorescence units (RFU), measured by Isoplexis CodePlex analysis] in relation to OIL-induced tumor cell death (TCD) (n=17 patients; 13 appendiceal and 4 mesothelioma). (D) Individual expression of effector cytokines Granzyme B and INF-γ measured from the same co-culture media in (C). Data points in all box plots represent individual patients. Box plots present minimum, median, and maximum values. Statistical significance calculated for linear mixed models using post hoc Tukey method. Pearson correlation coefficients analyzed using T-test. *p<0.05 **p< 0.01 ***p< 0.001 ****p< 0.0001.

Article Snippet: CD8 + T cells were isolated using human CD8 microbeads kit (Miltenyi Biotec, USA) according to manufacturer’s protocol.

Techniques: Comparison, Single Cell, Produced, Expressing, Cytotoxicity Assay, Co-Culture Assay, Fluorescence

Journal: bioRxiv

Article Title: Multiplexed high-content imaging uncovers morphological diversity of lymphocyte activation and dysfunction

doi: 10.64898/2026.02.10.704860

Figure Lengend Snippet: A) Experimental design. B-C) UMAP embeddings of 9,556 cells (15 PCs) which are annotated by: B) state (expanded: blue, exhausted: green) or by C) Louvain cluster number (1-8, resolution= 0.8). D) Volcano plot displaying the effect size (x-axis) versus the –log10(p-adjusted) from modelling CD8 + T cell states with expanded cells as a reference. Each dot represents the mean of a feature set, with grey circles indicating no statistical dysregulation and otherwise, colors indicating channels (ER: light blue, DNA: green, tubulin: purple, lipids: teal, mitochondria: green, CD25: yellow, AreaShape: dark blue, Ki67: burgundy) and shapes indicating categories (AreaShape: large circle, Intensity: diamond, Texture: circle and cross, Granularity: triangle, Radial Distribution (Radial Dist.): star). E-H) Single-cell violin plots of the transformed and scaled features with the median and interquantile range for each status indicated in a coloured point and line: expanded (blue) and exhausted (green). Significant differences (p adj ≤ 0.05) are shown with an asterisk (*). The features are: E) cell_Children_mito_Count (mitochondria count, left) and cell_Intensity_MassDisplacement_mito (mitochondria mass displacement, right), F) lipids_AreaShape_Area (size of lipids), G) nucl_Texture_Variance_dna_3_00_256 (DNA texture) and H) cell_Intensity_IntegratedIntensity_tubulin (tubulin integrated intensity). I-K) Connected plots summarising each cluster (row): I) proportion plot of expanded (blue) and exhausted (green) cells J) Representative images (n= 4, cell closest to the cluster centroid and its three nearest neighbours, deconvoluted and max projected) showing mitochondria (pink), tubulin (orange) and lipids (white). Scale bar= 2.5μm. K) Marker plot where the size of the dot represents the percentage of significant feature sets per cluster (row) in each channel (column) and the color depicts the mean marker effect size from –1 (blue) to 0 (white) and +1 (red).

Article Snippet: CD8 + T cells were isolated from healthy donors using the REAlease CD8 MicroBead Kit (Miltenyi Biotec, #130-117-036) according to the manufacturer’s instructions.

Techniques: Single Cell, Transformation Assay, Marker

Journal: Journal of Nanobiotechnology

Article Title: CD8a antibody-functionalized biomimetic red blood cell membrane ectosomes delivering C646 reverse CD8⁺ T Cell exhaustion via H3K18la histone delactylation in gastric cardia adenocarcinoma

doi: 10.1186/s12951-025-03957-z

Figure Lengend Snippet: Bioinformatics Analysis Identifies Key Factors in Histone Lactylation Modification. Note: ( A ) Schematic workflow of bioinformatics analysis for identifying key factors; ( B ) Volcano plot of DEGs in tumor tissues and adjacent normal tissues from dataset GSE2685 (Normal = 8, Tumor = 22); ( C ) Heatmap showing the correlation of co-expression module genes with tumor and normal tissues, with each cell displaying the correlation coefficient and p -value; ( D ) Venn diagram illustrating the intersection of Blue module genes, DEGs, CD8 + T cell-related genes, and "Histone lactylation"-related genes; ( E ) Box plot of p300 differential expression; ( F ) The tSNE distribution map of EP300 in various cell types in the scRNA-seq data

Article Snippet: Human CD8+ T cells were isolated from peripheral blood mononuclear cells (hPBMCs, PCS-800–011, ATCC, USA) using the CD8 MicroBead Kit (130–045–201, Miltenyi Biotec, USA).

Techniques: Modification, Expressing, Quantitative Proteomics

Journal: Journal of Nanobiotechnology

Article Title: CD8a antibody-functionalized biomimetic red blood cell membrane ectosomes delivering C646 reverse CD8⁺ T Cell exhaustion via H3K18la histone delactylation in gastric cardia adenocarcinoma

doi: 10.1186/s12951-025-03957-z

Figure Lengend Snippet: p300 Regulates Histone Lactylation in CD8 + T Cells. Note: ( A ) Schematic representation of the experimental design, showing the workflow for detecting CD8 + T cells treated with lactate, p300 inhibitors, or activators. ( B–C ) WB analysis of PKla levels in CD8 + T cells over time (B) and under varying lactate concentrations ( C ). ( D–E ) WB analysis of the time-dependent ( D ) and dose-dependent ( E ) changes in H3K18la and H3K9la expression in CD8 + T cells following lactate treatment. *** p < 0.001, ** p < 0.01, and * p < 0.05 compared to the 0-h or untreated lactate group. ( F ) WB analysis of H3K18la and H3K9la expression in CD8 + T cells following p300 knockdown, activation, or inhibition. ( G ) ELISA detection of IFN-γ levels in the supernatant of CD8 + T cells across different treatment groups. (H) FCM analysis of GZMB expression in CD8 + T cells. ( I ) FCM analysis of CD8 + T cell proliferation. ( J ) LDH release assay showing the cytotoxic effects of CD8 + T cells on MKN-45 and SNU1 cells. In panels ( F–J ), * p < 0.05, ** p < 0.01, and *** p < 0.001 compared to the control group; # p < 0.05, ## p < 0.01, and ### p < 0.001 compared to the Lactate group; & p < 0.01 compared to the Lactate + sh-NC group. All cell-based experiments were performed in triplicate

Article Snippet: Human CD8+ T cells were isolated from peripheral blood mononuclear cells (hPBMCs, PCS-800–011, ATCC, USA) using the CD8 MicroBead Kit (130–045–201, Miltenyi Biotec, USA).

Techniques: Expressing, Knockdown, Activation Assay, Inhibition, Enzyme-linked Immunosorbent Assay, Lactate Dehydrogenase Assay, Control

Journal: Journal of Nanobiotechnology

Article Title: CD8a antibody-functionalized biomimetic red blood cell membrane ectosomes delivering C646 reverse CD8⁺ T Cell exhaustion via H3K18la histone delactylation in gastric cardia adenocarcinoma

doi: 10.1186/s12951-025-03957-z

Figure Lengend Snippet: Preparation and Characterization of NVEs. Note: ( A ) Schematic illustration of the construction of CD8a-NVEs@C646; ( B ) DLS analysis of nanoparticle sizes for NVEs, NVEs@C646, and CD8a-NVEs@C646; ( C ) Zeta potential measurements for NVEs, NVEs@C646, and CD8a-NVEs@C646, showing surface charge variations with different modifications; (D) PDI values of NVEs, NVEs@C646, and CD8a-NVEs@C646; ( E ) TEM images for NVEs, NVEs@C646, and CD8a-NVEs@C646; ( F ) Au-immunogold TEM images for NVEs, NVEs@C646, and CD8a-NVEs@C646 (scale bar = 200 nm); ( G–H ) Stability analysis of particle sizes for NVEs, NVEs@C646, and CD8a-NVEs@C646 after 12, 24, 48, and 72 h of incubation in PBS and 10% FBS; ( I ) SDS-PAGE analysis of membrane protein content for NVEs (2), NVEs@C646 (3), and CD8a-NVEs@C646 (4), with protein marker (1); ( J ) Encapsulation efficiency of NVEs@C646 and CD8a-NVEs@C646 at different C646 concentrations; ( K ) In vitro release profiles of C646 from NVEs@C646 and CD8a-NVEs@C646 over different time points. Experiments were repeated three times

Article Snippet: Human CD8+ T cells were isolated from peripheral blood mononuclear cells (hPBMCs, PCS-800–011, ATCC, USA) using the CD8 MicroBead Kit (130–045–201, Miltenyi Biotec, USA).

Techniques: Zeta Potential Analyzer, Incubation, SDS Page, Membrane, Marker, Encapsulation, In Vitro

Journal: Journal of Nanobiotechnology

Article Title: CD8a antibody-functionalized biomimetic red blood cell membrane ectosomes delivering C646 reverse CD8⁺ T Cell exhaustion via H3K18la histone delactylation in gastric cardia adenocarcinoma

doi: 10.1186/s12951-025-03957-z

Figure Lengend Snippet: In Vivo and In Vitro Biocompatibility and Targeting Validation of NVEs. Note: ( A ) Schematic illustration of in vitro targeting and biocompatibility experiments of NVEs; ( B ) Hemolysis analysis of NVEs, NVEs@C646, and CD8a-NVEs@C646 after incubation with red blood cells (PC: positive control); ( C ) CD8 + T cell viability assessed using the CCK-8 assay after 48-h incubation with varying concentrations of NVEs; ( D ) Confocal microscopy and quantitative analysis of NVEs@C646 and CD8a-NVEs@C646 uptake in CD8 + T cells (Scale bars = 25 μm); (E) FCM analysis of the uptake efficiency of NVEs@C646 and CD8a-NVEs@C646 in CD8 + T cells; (F) Schematic illustration of the subcutaneous tumor model in mice and in vivo injection of NVEs; ( G ) FCM analysis of the percentage of CM-Dil-labeled CD8 + T cells in blood, spleen, tumor tissue, and TdLNs at different time points post-injection (* p < 0.05 compared with the NVEs@C646 group); ( H ) FCM analysis of CD3 + CD8 − T cells binding with CM-Dil-labeled NVEs recovered from blood, spleen, tumor tissue, and TdLNs at different time points; ( I ) Quantification of CD8 + T cell numbers 48 h after NVEs injection. Cell-based experiments were performed in triplicate. Animal experiments included nine mice per group, with three mice per time point

Article Snippet: Human CD8+ T cells were isolated from peripheral blood mononuclear cells (hPBMCs, PCS-800–011, ATCC, USA) using the CD8 MicroBead Kit (130–045–201, Miltenyi Biotec, USA).

Techniques: In Vivo, In Vitro, Biomarker Discovery, Incubation, Positive Control, CCK-8 Assay, Confocal Microscopy, Injection, Labeling, Binding Assay

Journal: Journal of Nanobiotechnology

Article Title: CD8a antibody-functionalized biomimetic red blood cell membrane ectosomes delivering C646 reverse CD8⁺ T Cell exhaustion via H3K18la histone delactylation in gastric cardia adenocarcinoma

doi: 10.1186/s12951-025-03957-z

Figure Lengend Snippet: CD8a-NVEs@C646 Facilitates Histone Delactylation Modification in CD8 + T Cells. Note: ( A ) Schematic workflow of RNA-seq and ChIP-seq experiments for CD8 + T cells treated with CD8a-NVEs@C646; ( B–C ) ChIP-seq analysis showing signals at TSS regions in PBS-treated (n = 3) and CD8a-NVEs@C646-treated groups (n = 3); ( D ) RNA-seq volcano plot illustrating significantly upregulated and downregulated genes in the CD8a-NVEs@C646-treated group compared to PBS (n = 3); ( E ) Venn diagram of overlapping genes from ChIP-seq and RNA-seq analyses related to CD8 + T cells; ( F ) ChIP analysis of p300 and H3K18la enrichment at the PDCD1 promoter region; ( G ) The protein interaction between p300 and H3K18la was detected by co-IP assay; ( H ) RT-qPCR analysis of PDCD1 mRNA expression in CD8 + T cells following CD8a-NVEs@C646 treatment; ( I ) WB analysis of PDCD1 and H3K18la protein expression levels in CD8 + T cells treated with CD8a-NVEs@C646. * p < 0.05, *** p < 0.001 compared to the PBS or sh-NC group; experiments were conducted in triplicate

Article Snippet: Human CD8+ T cells were isolated from peripheral blood mononuclear cells (hPBMCs, PCS-800–011, ATCC, USA) using the CD8 MicroBead Kit (130–045–201, Miltenyi Biotec, USA).

Techniques: Modification, RNA Sequencing, ChIP-sequencing, Co-Immunoprecipitation Assay, Quantitative RT-PCR, Expressing

Journal: Journal of Nanobiotechnology

Article Title: CD8a antibody-functionalized biomimetic red blood cell membrane ectosomes delivering C646 reverse CD8⁺ T Cell exhaustion via H3K18la histone delactylation in gastric cardia adenocarcinoma

doi: 10.1186/s12951-025-03957-z

Figure Lengend Snippet: CD8a-NVEs@C646-Mediated Regulation of PDCD1 Enhances the Tumor-Killing Ability of CD8 + T Cells. Note: ( A ) Schematic diagram of the experimental design: CD8 + T cells were transduced with PDCD1 overexpression lentivirus and treated with CD8a-NVEs@C646, followed by co-culture with MKN-45/SNU1 cells; ( B ) RT-qPCR analysis of PDCD1 mRNA expression in CD8 + T cells across different groups; ( C ) WB analysis of PDCD1 and H3K18la protein expression in CD8 + T cells across different groups; ( D ) ELISA detection of IFN-γ levels in the supernatant of CD8 + T cells across different groups; ( E ) FCM analysis of GZMB expression in CD8 + T cells across different groups; ( F ) FCM analysis of CD8 + T cell proliferation activity; ( G ) LDH release assay to evaluate the cytotoxicity of CD8 + T cells on MKN-45 cells; ( H ) FCM analysis of apoptosis in MKN-45 cells across different groups. * indicates p < 0.05 compared to the PBS + vector group, *** indicates p < 0.001, # indicates p < 0.05 compared to the CD8a-NVEs@C646 + vector group, ### indicates p < 0.001. All cellular experiments were repeated three times

Article Snippet: Human CD8+ T cells were isolated from peripheral blood mononuclear cells (hPBMCs, PCS-800–011, ATCC, USA) using the CD8 MicroBead Kit (130–045–201, Miltenyi Biotec, USA).

Techniques: Transduction, Over Expression, Co-Culture Assay, Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay, Activity Assay, Lactate Dehydrogenase Assay, Plasmid Preparation

Journal: Journal of Nanobiotechnology

Article Title: CD8a antibody-functionalized biomimetic red blood cell membrane ectosomes delivering C646 reverse CD8⁺ T Cell exhaustion via H3K18la histone delactylation in gastric cardia adenocarcinoma

doi: 10.1186/s12951-025-03957-z

Figure Lengend Snippet: CD8a-NVEs@C646 Enhances CD8 + T Cell-Mediated Anti-Tumor Immunity. Note: ( A ) Schematic diagram of the experimental design, illustrating the establishment of the GCA orthotopic transplantation tumor model and treatment regimen; ( B ) In vivo fluorescence imaging of tumors in mice from different treatment groups and quantitative analysis of signal intensity over time; ( C ) Photographic images of tumor tissues from different treatment groups; ( D ) Tumor weight statistics for each group; ( E–F ) Immunohistochemistry and TUNEL staining showing cell proliferation and apoptosis in tumor tissues from each group (Scale bars = 50 μm); ( G ) Quantification of the proportion of infiltrating CD8 + T cells in tumor tissues from each group; ( H-J ) Expression levels of IFN-γ, TNFα, and GZMB in CD8 + T cells within tumor tissues from each group; ( K ) Proportion of PD-1 + CD8 + T cells in tumor tissues from each group. Six mice per group; * indicates p < 0.05 compared to the NS group, and # indicates p < 0.05 compared to the αPD-1 group

Article Snippet: Human CD8+ T cells were isolated from peripheral blood mononuclear cells (hPBMCs, PCS-800–011, ATCC, USA) using the CD8 MicroBead Kit (130–045–201, Miltenyi Biotec, USA).

Techniques: Transplantation Assay, In Vivo, Fluorescence, Imaging, Immunohistochemistry, TUNEL Assay, Staining, Expressing

Journal: Nature Communications

Article Title: Meta-unstable mRNAs in activated CD8 + T cells are defined by interlinked AU-rich elements and m 6 A mRNA methylation

doi: 10.1038/s41467-025-67762-w

Figure Lengend Snippet: A The miCLIP workflow for identification of high-confidence crosslinks: (1) primary human CD8 + T cells subjected to in vitro activation with aCD3/aCD28 beads; (2) RNA enrichment and experimental controls, including m 6 A immunoprecipitation without UV irradiation (noUV) and input RNA samples; (3) SDS‒PAGE purification of RNA fragments crosslinked to the anti-m 6 A antibody followed by RNA isolation; (4) library preparation; (5) iCLIP analysis for determination of uniquely mapped crosslinks; (6) determination of peaks, i.e., regions with high density of crosslinks; (7) ‘positionally enriched k-mer analysis’ (PEKA) to extract sequences in high-confidence (‘thresholded’) miCLIP crosslinks. B Hierarchical clustering of 5-mers based on standardised PEKA scores at ‘thresholded sites’; here, crosslinks and Clippy peaks were used for PEKA. The median PEKA score of each miCLIP or Input group (noAct, Day1 or Day5) was calculated for all 5-mers, and the unique sets of the 20 most enriched and variant 5-mers were plotted on the heatmap. C Metagene distribution of GLORI-determined m 6 A counts at miCLIP sites (top) and number-matched control sites randomly selected from the 3’UTR (bottom), centred on RRACH, WWAWW and CRACH pentamers; GLORI counts show the effect of the METTL3 inhibitor at each position, and represent the average across two CD8 + T cell replicates. D Metagene distribution of GLORI-determined m 6 A counts centred on miCLIP-ARE sites that contain nearby RRACH motifs (left) or lack RRACH motifs (right), showing the effect of the METTL3 inhibitor. Stratification by distance highlights the spatial relationship between m 6 A deposition and RRACH proximity. The WWAWW + RRACH sites refer to miCLIP-ARE sites containing at least one RRACH motif within ≤ 50nt. E Density plot showing the motif-specific distribution of PEKA crosslinks in the miCLIP and Input samples around scaled mRNA regions. For all panels, CD8 + T cells from healthy donors were isolated; not activated (noAct; n = 4), Day1 after activation (Day1; n = 5), Day5 after activation (Day5; n = 4), Mock ( n = 12), noUV ( n = 3). Panel ( A ) was created in BioRender. Foskolou, I. (2026) https://BioRender.com/ h18sinc. Source data are provided as a Source Data file.

Article Snippet: Peripheral blood mononuclear cells (PBMCs) were thawed, and CD8 + T cells were isolated using a CD8+ isolation kit (CD8 + MicroBeads, 130-045-201, Miltenyi biotec).

Techniques: In Vitro, Activation Assay, Immunoprecipitation, Irradiation, Purification, Isolation, Variant Assay, Control

Journal: Nature Communications

Article Title: Meta-unstable mRNAs in activated CD8 + T cells are defined by interlinked AU-rich elements and m 6 A mRNA methylation

doi: 10.1038/s41467-025-67762-w

Figure Lengend Snippet: A Actinomycin D experiments showing % of remaining mRNA in CD8⁺ T cells treated with DMSO or METTL3inhibitor. Mean ± SD; n = 3 (one donor provided material for 0,2 h). Mixed-effects model (REML), p -values indicate treatment effects. Donors were processed in parallel. Representative results from three independent experiments ( IL7R , TNF ). B mRNA levels IL7R ( n = 5), CCL4 ( n = 5), CD69 ( n = 4), CD28 ( n = 4), TCF7 ( n = 3), ZFP36L1 ( n = 3), TNF ( n = 4), IFNG ( n = 4) in CD8⁺ T cells treated with METTL3inhibitor. Fold change relative to DMSO (grey line). Reference gene 18S. Mean ± SD; paired two-tailed t tests on ΔCt values. For TNF , one Grubbs-identified outlier is displayed but excluded from statistics. C , D Representative flow cytometry plots illustrating IL7Rα gating ( C ) and quantification of IL7Rα⁺CD8⁺ T cells ( D ) ( n = 10). Paired two-tailed t tests. E , F Representative histogram ( E ) and quantification ( F ) of IL7Rα median fluorescence intensity (MFI) in IL7Rα⁺ cells treated with METTL3inhibitor relative to DMSO (grey line) ( n = 10). Mean ± SD; one-sample two-tailed t test. G GFP MFI in T cells transduced with GFP-3’UTR constructs (S3H) after treatment with FTO inhibitor followed by PMA/ionomycin activation relative to DMSO ( n = 3). One-way ordinary ANOVA. H , I IFNγ (H) and TNFα (I) protein levels measured by ELISA in DMSO (0 μM) or FTOinhibitor treated cells ( n = 3). RM one-way ANOVA. J , K Representative amplification curves (ΔRn) and Ct quantification for reference ( J ) and miCLIP-ARE ( K ) amplicons in control (grey) and METTL3-KO (pink) samples ( n = 3). Mean ± SD. L Fold-change expression (ΔΔCt) for the miCLIP-ARE site in METTL3-KO and FTO-KO cells relative to control (grey line). Mean ± SEM; unpaired two-tailed t test comparing METTL3-KO and FTO-KO; paired t test for control vs METTL3-KO on ΔCt values; ( n = 3). M – P mRNA levels ( M ) and GFP protein levels ( N – P ) in CD8 + T cells transduced with GFP-3′UTR constructs (S3O). mRNA levels normalised to 3′UTR- TNF_ WT (grey line) ( n = 10); one-way ANOVA with Holm–Šídák correction ( M ). Representative plots of resting or PMA/ionomycin-activated cells ( N ). GFP MFI quantification in activated cells ( O , P ) ( n = 15); paired two-tailed t tests. Box-and-whisker plots (min–max, median, 25th–75th percentiles). All n values reflect distinct donors. Source data are provided as a Source Data file.

Article Snippet: Peripheral blood mononuclear cells (PBMCs) were thawed, and CD8 + T cells were isolated using a CD8+ isolation kit (CD8 + MicroBeads, 130-045-201, Miltenyi biotec).

Techniques: Two Tailed Test, Flow Cytometry, Fluorescence, Transduction, Construct, Activation Assay, Enzyme-linked Immunosorbent Assay, Amplification, Control, Expressing, Whisker Assay