spectral flow cytometry analysis  (Cytek Biosciences)

Journal: ACS applied materials & interfaces

Article Title: Manganese-Loaded pH-Responsive DNA Hydrogels Enable Tg-Guided Thyroid Tumor Targeted Magnetic Resonance Imaging.

doi: 10.1021/acsami.4c19676

Figure Lengend Snippet: Figure 3. In vitro stability and cytotoxicity of M-TDH. (a) Tyndall effect showed good stability of M-TDH in saline with 10% FBS. (b) Cell viability after 24 h of incubation with PBS, TDH, M-TDH, and MnCl2, detected by CCK-8. (c, d) Cell apoptosis rates of DTC cells detected by flow cytometry. (e, f) Live/dead cell staining images and corresponding quantitative comparison of TPC1 and K1 cells after various treatments (scale bar 100 μm). (g, h) Hematological parameters of M-TDH detected by hemolysis experiments and corresponding quantitative measurements.

Article Snippet: The quantification of cellular uptake was obtained by full spectrum analysis flow cytometry (ID7000 Sony, Japan).

Techniques: In Vitro, Saline, Incubation, CCK-8 Assay, Flow Cytometry, Staining, Comparison

Journal: ACS applied materials & interfaces

Article Title: Manganese-Loaded pH-Responsive DNA Hydrogels Enable Tg-Guided Thyroid Tumor Targeted Magnetic Resonance Imaging.

doi: 10.1021/acsami.4c19676

Figure Lengend Snippet: Figure 5. Uptake efficiency and aptamer-mediated combination. (a, b) The uptake efficiency of M-TDH or TDH by K1 and TPC-1 cells detected by flow cytometry. (c) The in situ targeting of the nanohydrogel in frozen thyroid tissue sections. The white box shows the enlarged area, Cy5- TDH shows a red fluorescence signal, and a green fluorescence signal indicates the Tg distribution. (d) Nucleic acid−protein binding assay used to compare the combination between DH or TDH and endogenous Tg protein in the cellular environment.

Article Snippet: The quantification of cellular uptake was obtained by full spectrum analysis flow cytometry (ID7000 Sony, Japan).

Techniques: Flow Cytometry, In Situ, Fluorescence, Protein Binding

Journal: bioRxiv

Article Title: Characterization of adult human skeletal cells in different tissues reveals a CD90+CD34+ periosteal stem cell population

doi: 10.1101/2022.12.05.519079

Figure Lengend Snippet:

Article Snippet: Spectral flow cytometry analysis was performed on a Cytek Northern Lights instrument with three lasers.

Techniques: Flow Cytometry

Journal: Journal for Immunotherapy of Cancer

Article Title: Selective targeting of GARP-LTGFβ axis in the tumor microenvironment augments PD-1 blockade via enhancing CD8 + T cell antitumor immunity

doi: 10.1136/jitc-2022-005433

Figure Lengend Snippet: In vitro characterization of anti-GARP antibody PIIO-1. (A) GARP expression on human regulatory T cells and platelets was evaluated by flow cytometry after staining with PIIO-1 at 10 µg/mL. (B) 293 FT cell line was transfected with empty vector (EV), human GARP (hGARP)-expressing vector only or co-transfected with hGARP and latent TGFβ1 expression vectors. GARP expression on indicated cell line was detected by flow cytometry after staining with PIIO-1 at 10 µg/mL. (C) Human GARP sequence was replaced by murine GARP according to the schematic diagram to generate the chimeric constructs of human and murine GARP that were tagged with HA (hemagglutinin) epitope. Transfection efficiency was determined using anti-HA antibody. All constructs were transfected into 293 FT cells. (D) Crystal structure of the GARP (green)-LTGFβ (gray) complex (PDB DOI: 10.2210/pdb6GFF/pdb). The region of PIIO-1 recognition is orange and the residues interacting with LTGFβ are cyan. LTGFβ occludes approximately 30% of the potential antibody binding site and may sterically or allosterically restrict access of the antibody to GARP in the LTGFβ-complexed state. Modeling was carried out using Pymol. (E) Jurkat cell line, made to overexpress hGARP, was incubated with LTGFβ1 along with mIgG1 or PIIO-1 at indicated concentration for 30 min at 37℃. Human LAP expression was detected by flow cytometry. All data are representative of 2–6 independent experiments. GARP, Glycoprotein-A repetitions predominant; LAP, latency-associated peptide.

Article Snippet: Antibody staining and high dimensional spectral flow cytometry analysis (Cytek) were performed as detailed previously.

Techniques: In Vitro, Expressing, Flow Cytometry, Staining, Transfection, Plasmid Preparation, Sequencing, Construct, Binding Assay, Incubation, Concentration Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Selective targeting of GARP-LTGFβ axis in the tumor microenvironment augments PD-1 blockade via enhancing CD8 + T cell antitumor immunity

doi: 10.1136/jitc-2022-005433

Figure Lengend Snippet: PIIO-1 monotherapy modulates CD8 + T cells in the TME and confers protection against cancer in hLRRC32 KI mice. (A) 1×10 5 MB-49 cells were injected s.c. on the right flank of hLRRC32 KI male mice. PIIO-1 or isotype control (ISO) was administered (200 µg/mouse, i.p.) every 3 days for a total of four treatments starting on day 4. Shown is one representative tumor growth curve. (B) 1×10 5 MB-49 cells were injected s.c. on the right flank of hLRRC32 KI male mice. PIIO-1 was delivered (200 µg/mouse, i.p.) on days 6 and 9. Tumors were collected on day 10 and tumor-infiltrating leucocytes were stained and analyzed by flow cytometry. Frequency of CD8 + T cells as a proportion of live CD45 + lymphocytes is shown (left). A similar experiment was performed wherein mice were treated with PIIO-1 for a total of 6 treatments starting on day 6. CD8 + TILs were quantified on day 22 (right). (C) Frequency of total Tregs (CD25 + Foxp3 + ) (left) and CTLA4 + VISTA + Tregs (right) in tumor-infiltrating CD4 + T cells in the indicated treatment groups. (D) Differential expression analysis of cluster frequency of CD8 + TILs between ISO and PIIO-1 treated TILs. UMAP dimension reduction of tumor-infiltrating CD8 + T cells from B after staining with 33 markers and high dimensional spectral flow cytometry analysis. Data shown is gated on live CD45 + CD3 + CD8 + T cells, subsampled on 5000 cells per sample. Unsupervised clustering analysis was done using FlowSOM algorithm with an elbow method approach for cluster number determination. (E) Heatmap of D showing the relative expression levels of indicated markers by each cluster. (A–E) n=4–6 mice per group. (F) Differential expression analysis of cytokine production by CD8 + TILs between ISO and PIIO-1 treated tumors. 1×10 5 MB-49 cells were injected s.c. on the right flank of hLRRC32 KI male mice. PIIO-1 or IOS was administered every 3 days for a total of 4 treatments starting on day 5. Tumors were collected on day 17. Intracellular stain for 17 cytokine panel was done, followed by spectral flow cytometry and analysis of CD45 + CD3 + CD8 + T cells. (G) Cytokine level in panel (F) indicated by heatmap showing expression intensity of cytokines by each CD8 + T cell cluster. Tumor curve analysis was performed using repeated measures two-way analysis of variance. Cluster differences were measured by two-tailed Student’s t test. Data are represented as mean±SEM. *P<0.05, **p<0.01, ****p<0.0001.

Article Snippet: Antibody staining and high dimensional spectral flow cytometry analysis (Cytek) were performed as detailed previously.

Techniques: Injection, Control, Staining, Flow Cytometry, Expressing, Two Tailed Test

Journal: Journal for Immunotherapy of Cancer

Article Title: Selective targeting of GARP-LTGFβ axis in the tumor microenvironment augments PD-1 blockade via enhancing CD8 + T cell antitumor immunity

doi: 10.1136/jitc-2022-005433

Figure Lengend Snippet: PIIO-1 attenuates canonical TGFβ signaling pathway in tumor-infiltrating immune cells and rejuvenates antitumor immunity in hLRRC32 KI mice. (A) 1×10 5 MB-49 cells were injected s.c. on the right flank of hLRRC32 KI male mice. Humanized PIIO-1 (200 µg/mouse, i.p.) were administered on days 18 and 20. Tumors were collected on day 21. TILs were isolated and stained for intracellular pSMAD2/3 with indicated cell linage markers, followed by flow cytometry analysis. (B) Quantification of panel A. (C–E) 1×10 5 MB-49 cells were injected s.c. on the right flank of hLRRC32 KI male mice. Humanized PIIO-1 (200 µg/mouse, i.p.) was delivered on days 6 and 9. Tumors were collected on day 10. Single cell suspension and RNA isolation were prepared, and then subjected to bulk RNA sequencing. (C) Volcano plot of transcript expression. Differential gene expression was shown in red (up) or blue (down). Representative transcripts such as Ccl3, Ccl9, Cxcl14, Cxcl15 , Il6 and Tnfrsf25 were indicated. (D) GSEA of differential expression genes between tumors treated with PBS and PIIO-1. (E) Comparison of TILs between PBS and PIIO-1 treated tumors based on deconvolution of bulk RNA sequencing data. Data were performed using two-tailed Student’s t-test, data in (B) represented mean±SEM. *p<0.05, **p<0.01.

Article Snippet: Antibody staining and high dimensional spectral flow cytometry analysis (Cytek) were performed as detailed previously.

Techniques: Injection, Isolation, Staining, Flow Cytometry, Suspension, RNA Sequencing Assay, Expressing, Comparison, Two Tailed Test