Journal: Cell communication and signaling : CCS

Article Title: Tumorous IRE1α facilitates CD8 + T cells-dependent anti-tumor immunity and improves immunotherapy efficacy in melanoma.

doi: 10.1186/s12964-024-01470-8

Figure Lengend Snippet: Fig. 2 Tumorous IRE1α facilitates anti-tumor immunity in a CD8+T cells-dependent manner. A Schema of the treatment in C57BL/6 mice bearing B16F10 tumors received HA15 treatment as indicated. Tumor burdens, weights and volumes in each group were calculated and displayed in B and C. D Representative flow cytometry data and summary plots of the frequency of CD3+CD45+, CD8+CD3+, CD11c+CD45+, Foxp3+CD4+ and CD8+T-cells evaluated for expression of Granzyme B and IFN-γ in tumor from xenografts with indicated treatment. E Immunofluorescence staining of XBP1s or CD8α in B16F10 xenografts with or without the treatment of HA15. Scale bar, 50 μm. F-H Scheme representing the experimental procedure (F), tumor burdens, tumor weight (G), and tumor volume (H) of C57BL/6 mice injected subcutaneously with B16F10 tumors with treatment of HA15 and αCD8 depleting antibodies either alone or in combination. Symbols of one dot indicates one mouse, and the error bars are mean with ± SD (n = 4). Two-tailed Student’s t-test (*p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant)

Article Snippet: Heat-mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) was performed, the sections were blocked with 10% goat serum in PBS for 1 h at room temperature and then stained overnight at 4 °C with rabbit pAb to XBP1s (1:100, 24868-1-AP, proteintech), rabbit pAb to Annexin A1 (1:50, 21990-1-AP, proteintech), rabbit pAb to Calreticulin (1:50, 27298-1-AP, proteintech), rabbit pAb to HMGB1 (1:50, 10829-1-AP, proteintech), rabbit mAb to p-MLKL (Ser345) (1:500, 37,333, CST), mouse mAb to Foxp3 (1:50, 65089-1-Ig, proteintech), CoraLite® Plus 488 Rat mAb to CD4 (1:50, CL488-65141, proteintech), rabbit pAb to Granzyme B (1:50, 13588-1- AP, proteintech), rabbit pAb to IFN-γ (1:50, 105,995-T08, Sinobiological) or rat mAb to CD8α (1:50, 65069-1-Ig, proteintech) antibody.

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

Journal: Cell communication and signaling : CCS

Article Title: Tumorous IRE1α facilitates CD8 + T cells-dependent anti-tumor immunity and improves immunotherapy efficacy in melanoma.

doi: 10.1186/s12964-024-01470-8

Figure Lengend Snippet: Fig. 6 ER stress inducer enhances the anti-tumor activity of anti-PD-1 antibody in vivo. A Schema of the treatment in C57BL/6 mice bearing B16F10 tumors received HA15 with or without anti-PD-1 antibody combination treatment as indicated. B Images of isolated tumors from mice that received indicated treatment. Tumor volumes and weights in each group were calculated and displayed in C and D. E-H Representative flow cytometry data and summary plots of the frequency of CD3+CD45+, CD8+CD3+, F4/80+CD11b+CD45+, Foxp3+CD25+CD4+ and CD8+T-cells evaluated for expression of Granzyme B and IFN-γ in tumor from xenografts with indicated treatment. Symbols of one dot indicates one mouse, and the error bars are mean with ± S.D (n = 4). Two-tailed Student’s t-test (*p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant)

Article Snippet: Heat-mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) was performed, the sections were blocked with 10% goat serum in PBS for 1 h at room temperature and then stained overnight at 4 °C with rabbit pAb to XBP1s (1:100, 24868-1-AP, proteintech), rabbit pAb to Annexin A1 (1:50, 21990-1-AP, proteintech), rabbit pAb to Calreticulin (1:50, 27298-1-AP, proteintech), rabbit pAb to HMGB1 (1:50, 10829-1-AP, proteintech), rabbit mAb to p-MLKL (Ser345) (1:500, 37,333, CST), mouse mAb to Foxp3 (1:50, 65089-1-Ig, proteintech), CoraLite® Plus 488 Rat mAb to CD4 (1:50, CL488-65141, proteintech), rabbit pAb to Granzyme B (1:50, 13588-1- AP, proteintech), rabbit pAb to IFN-γ (1:50, 105,995-T08, Sinobiological) or rat mAb to CD8α (1:50, 65069-1-Ig, proteintech) antibody.

Techniques: Activity Assay, In Vivo, Isolation, Flow Cytometry, Expressing, Two Tailed Test

Journal: PLoS ONE

Article Title: Neutrophil Gelatinase-Associated Lipocalin Increases HLA-G + /FoxP3 + T-Regulatory Cell Population in an In Vitro Model of PBMC

doi: 10.1371/journal.pone.0089497

Figure Lengend Snippet: A). The percentage of CD4 + /CD25 + /FoxP3 + cells in PHA-activated PBMCs after stimulation with increasing concentrations of NGAL: Enterobactin:Iron (40 ng/ml, 80 ng/ml, 160 ng/ml, and 320 ng/ml), raised in a dose dependent manner. Data are representative of 8 independent experiments. The number in each quadrant represents the percentage of CD25 + /FoxP3 + cells on gated CD4 + cells of the total population. B) Data are expressed as percentages of CD4 + /CD25 + /FoxP3 + cells. Means ± SD; n = 8. *p<0.05.

Article Snippet: After permeabilization, cells were incubated for 30 minutes in the dark in the refrigerator (2–8°C) with an anti-FoxP3-PE antibody (Miltenyi, Bergish Gladbach, Germany).

Techniques:

Journal: PLoS ONE

Article Title: Neutrophil Gelatinase-Associated Lipocalin Increases HLA-G + /FoxP3 + T-Regulatory Cell Population in an In Vitro Model of PBMC

doi: 10.1371/journal.pone.0089497

Figure Lengend Snippet: Flow cytometry analysis of CD25 + /FoxP3 + expression on CD4 + cells in PHA-activated PBMCs. Iron treatment (without NGAL) did not increase the percentage of CD25 + /FoxP3 + cells. Treatment with NGAL:enterobactin complex (without iron) reduced the percentage of CD25 + /FoxP3 + cells in contrast to stimulation with NGAL:Enterobactin:Iron. Incubation with anti-NGAL antibody reduced the percentage of CD25 + /FoxP3 + cells. Data are representative of 8 independent experiments. The number in each quadrant represents the percentage of CD25+foxP3 cells on gated CD4 + cells of the total population. B) Data are expressed as percentages of CD4+/CD25+/FoxP3+ cells. Means ± SD; n = 8. * p<0.05.

Article Snippet: After permeabilization, cells were incubated for 30 minutes in the dark in the refrigerator (2–8°C) with an anti-FoxP3-PE antibody (Miltenyi, Bergish Gladbach, Germany).

Techniques: Flow Cytometry, Expressing, Incubation

Journal: PLoS ONE

Article Title: Neutrophil Gelatinase-Associated Lipocalin Increases HLA-G + /FoxP3 + T-Regulatory Cell Population in an In Vitro Model of PBMC

doi: 10.1371/journal.pone.0089497

Figure Lengend Snippet: Flow cytometry analysis of HLA-G + /FoxP3 + expression on CD4 + cell PHA-activated PBMCs. PBMCs were treated with increasing concentrations of NGAL:Enterobactin:Iron (40 ng/ml, 80 ng/ml, 160 ng/ml, and 320 ng/ml). A dose-dependent raise in the percentage of HLA-G + /FoxP3 + cells was evident. Data are representative of 8 independent experiments. The number in each quadrant represents the percentage of HLA-G + /FoxP3 + cells on gated CD4 + cells of the total population. B) Data are expressed as percentages of CD4 + /HLA-G + /FoxP3 + cells. Means ± SD; n = 8. * p<0.05.

Article Snippet: After permeabilization, cells were incubated for 30 minutes in the dark in the refrigerator (2–8°C) with an anti-FoxP3-PE antibody (Miltenyi, Bergish Gladbach, Germany).

Techniques: Flow Cytometry, Expressing

Journal: Current Protocols

Article Title: Do more with Less: Improving High Parameter Cytometry Through Overnight Staining

doi: 10.1002/cpz1.589

Figure Lengend Snippet: Intracellular and intranuclear staining can be improved with extended incubation times. (A) Representative staining obtained in 30 min versus 16 hr. (B) Stain indices for Foxp3 and IL‐2. Mouse splenocytes were stimulated and stained for intracellular cytokines as described in the Methods section. Data were acquired on a Cytek Aurora Spectral Cytometer.

Article Snippet: Anti‐mouse Foxp3 APC , REA788 , 1:200 , 5 , Miltenyi Biotec , 130‐111‐601.

Techniques: Staining, Incubation, Cytometry

Journal: Current Protocols

Article Title: Do more with Less: Improving High Parameter Cytometry Through Overnight Staining

doi: 10.1002/cpz1.589

Figure Lengend Snippet: Increasing incubation time reduces batch effects. (A) Representative staining and MFI of SIGLEC‐8 on CD45 + SSC hi CD16 ‐ cells from the same donor over 3 independent experiments. (B) Representative staining and MFI of CD123 on CD45 + SSC lo CD3 − CD19 − CD14 − CD16 − cells from the same donor over 3 independent experiments. (C) Human whole blood immunophenotyping data from the same donor over three independent experiments, stained for 30 min or 16 hr. tSNE plots were generated using the parameters CD45, SSC‐A, CD4, CD8, CD127, CD16, CD19, CD3, CD123, CD20, CD25, Fcer1a, CD11c, SIGLEC‐8, CD56, CD14, and HLA‐DR. Data were acquired on a BD LSRFortessa cytometer. FlowSOM clusters are shown in a colored overlay. (D) Cross entropy distances between samples stained for 30 min or 16 hr. (E) Mouse data from four experiments over the course of two years. Data were acquired on a BD FACSymphony A5 cytometer. tSNE plots were generated using the parameters CD4, CD8, Foxp3, CD103, Neuropilin, CD44, CD62L, Ki67, ICOS, PD‐1, CTLA‐4, CD25, KLRG1, CD69, ST2, and Helios on CD3 + T cells. FlowSOM clusters are shown in a colored overlay. (F) Cross entropy distances between mouse samples (intra‐batch variation) or batches (inter‐batch variation). Significance was tested by unpaired t‐test.

Article Snippet: Anti‐mouse Foxp3 APC , REA788 , 1:200 , 5 , Miltenyi Biotec , 130‐111‐601.

Techniques: Incubation, Staining, Generated, Cytometry

Journal: Current Protocols

Article Title: Do more with Less: Improving High Parameter Cytometry Through Overnight Staining

doi: 10.1002/cpz1.589

Figure Lengend Snippet: Titration is essential to maximize sensitivity. (A) Titration of CD3‐Spark Blue 550. (B) Intracellular overnight staining for PD‐1 on viable CD4 + CD3 + T cells at the indicated dilutions on cells fixed and permeabilized with the eBioscience Foxp3 Fix/Perm kit.

Article Snippet: Anti‐mouse Foxp3 APC , REA788 , 1:200 , 5 , Miltenyi Biotec , 130‐111‐601.

Techniques: Titration, Staining

Journal: Current Protocols

Article Title: Do more with Less: Improving High Parameter Cytometry Through Overnight Staining

doi: 10.1002/cpz1.589

Figure Lengend Snippet: Controls confirm specificity is maintained with overnight staining. (A) IL‐2 staining on WT or IL‐2‐deficient mouse CD4 + T cells. (B) pSTAT5 and Foxp3 staining on mouse CD4 + T cells with or without IL‐2 stimulation.

Article Snippet: Anti‐mouse Foxp3 APC , REA788 , 1:200 , 5 , Miltenyi Biotec , 130‐111‐601.

Techniques: Staining

Journal: Current Protocols

Article Title: Do more with Less: Improving High Parameter Cytometry Through Overnight Staining

doi: 10.1002/cpz1.589

Figure Lengend Snippet: Reagents Used for This Study

Article Snippet: Anti‐mouse Foxp3 APC , REA788 , 1:200 , 5 , Miltenyi Biotec , 130‐111‐601.

Techniques: Concentration Assay

Journal: Molecular Cancer

Article Title: PhiC31/PiggyBac modified stromal stem cells: effect of interferon γ and/or tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) on murine melanoma

doi: 10.1186/1476-4598-13-255

Figure Lengend Snippet: Determination of immune effectors following stem cell therapy. A) Infiltration of immune cells into the melanoma lung tumors. The lung tumors stained with monoclonal antibodies specific for mouse CD4, FOXP3, IL2 and CD8. Only respective images of CD4+, FOXP3+ and IL2+ cells from control samples are shown as there was no statistically significant difference between groups. Black arrows denote FOXP3+ and Il2+ cells. Results show significant infiltration of CD8+ cell into the melanoma lung tumors after injection of IFNγ-expressing ADSCs. Scale bars =50 μm. B) As shown in the bar graphs, mean number of CD8+ cells significantly increased in metastatic melanoma tumors of mice treated with IFNγ-ADSCs in a statistically meaningful manner. N =6; *** P < .001. C and D) Representative FACS plots for analysis of systemic CD4+ and regulatory T cells in peripheral blood obtained from the sub-ocular region of mice with established melanoma metastases. In addition, blood samples were taken from normal mice to measure normal number of CD4+ and Tregs. C) Left plot is representative FACS plot showing two distinct populations of PBMCs: lymphocytes and monocytes. Right plot is representative FACS plot indicating CD4 positive PBMCs. D) CD4+ lymphocytes were analyzed for expression of CD25 and FOXP3 by flow cytometry. Representative plots indicating analysis of Tregs in Normal, Control, EGFP-ADSCs and IFNγ-ADSCs. E) The percentage of CD4+ cells indicated a statistically significant decreased systemic level of CD4+ cells in ADSC-injected groups. The plot also shows a statistically significant decrease in the percentage of Tregs in the peripheral blood of mice with melanoma metastases treated with IFNγ-producing ADSCs. This percentage is comparable with systemic regulatory T cells of normal mice ( P = .109; IFNγ-ADSCs compared to normal mice). N =6; *** P < .001, ** P < .01, * P < .05.

Article Snippet: Subsequently, the PBMCs were incubated with a PE-conjugated monoclonal anti-mouse Foxp3 Ab, following the staining protocol provided by Miltenyi Biotec.

Techniques: Staining, Bioprocessing, Control, Injection, Expressing, Flow Cytometry