cd11b conjugated pe vio770  (Miltenyi Biotec)

Journal: bioRxiv

Article Title: Awakening intracellular immunity for functional HIV cure

doi: 10.64898/2026.01.04.697570

Figure Lengend Snippet: a , RAG1/RAG2 expression in HIV-infected human CD4⁺ T cells following DOS treatment (single dose, 4h), showing rapid induction and complete resolution within 72 hours of treatment. MFI, mean fluorescence intensity (RAG1, n = 6 donors; RAG2, n = 5 donors). In some experiments, the T cells were pre-treated with MG-132 (1mM) prior to DOS treatment ( top right ). b , Reversible DNA-damage signalling assessed by γH2AX foci formation following rejuvenation driven HIV eradication, with resolution over time (96h) and no evidence of persistent genomic stress ( n = 6 donors). c , Stem-like T cell induction among cured T cells (CD4 + CD45RA + CD27 + CD28 + CCR7 + , CD62L + , CD95 + ) after a one-week culture and repeated dose treatment throughout (dose escalation). Data are from n = 6 donors. d , Cell viability (Annexin V) show no increase in cell death following DOS exposure, indicating that HIV clearance is not attributable to cytotoxicity or clonal selection ( n = 3 donors). Similar RAG/ γH2AX resolution was observed following repeated (dose escalation) DOS treatment in one-week cultures. In a - d , a paired Student’s t-test. Data are shown as mean ± s.e.m. *** P < 0.001; **** P < 0.0001; ns, not significant.

Article Snippet: In some experiments, cells were evaluated for H2AX (Cell Signaling, 2595S; 1:100) and stem like reprogramming with antibodies to CD4 (PerCP5.5, BioLegend, 557956; 1:100), CCR7 (Brillant Violet 421, Biolegend, 100540; 1:100); CD27 (PerCP-Vio 700, Miltenyi; 130-120-037; 1:100); CD28 (PerCP/Cy5.5, BioLegend, 302922; 1:100); CD45RA (BV510, BioLegend, 304142; 1:100); CD62L (PE-Vio 770; Miltenyi; 130-113-621; 1:100) and CD95 (FITC, Miltenyi, 130-122-950; 1:100).

Techniques: Expressing, Infection, Fluorescence, Selection

Journal: bioRxiv

Article Title: Decoy Antibodies Block Extracellular HSP70, Prevent Self Signaling and Inhibit Melanoma Cell Survival

doi: 10.64898/2025.12.29.696857

Figure Lengend Snippet: A. Schematic representation of the melanosome-immunization and melanoma challenge protocol. C57BL/6 mice were immunized with 1 µg melanosomes or PBS on day 0, followed by booster immunizations with 10 µg of melanosomes or PBS on days 20 and 41. On day 60, mice were challenged with a subcutaneous injection of 100,000 B16 melanoma cells. Blood samples were collected on days 19, 40, and 59 (n=9 per group). B. Kaplan-Meier survival curves comparing melanosome-immunized mice (blue, n=9) and PBS-immunized mice (black, n=9) after B16 melanoma challenge; the experiment was repeated 2 times. C. End point day for each mouse. Time to reach the tumor growth limit for individual mice in groups immunized melanosomes (n=9) and PBS (n=9). D. Tumor volume quantification (in mm³) over time in melanosome-immunized (blue circles, n=9) and PBS-immunized mice (white circles, n=9). Tumor volume was calculated as follows: V = 0.5 × Length × Width 2 . E. Serum IgG responses to melanosomes in melanosome-immunized mice (left, n=9) and in PBS-immunized controls (right, n=9); as measured by ELISA, presenting the raw O.D.650 nm at three time points: day 19 (#1), day 40 (#2), and day 59 (#3); the experiment was repeated 3 times. F-I. Flow cytometry plots of cell populations in the spleens of one representative melanosome-immunized and PBS-immunized mouse. The frequencies of the gated populations of all (n=9) mice in each group are presented in the right panels. The immune populations include- F: B cells (B220 + ), G: GC B cells (B220 + CD95 (FAS) + GL7 + ), H: CD4 + T cells (CD3 + CD4 + ), and I: CD8 + T cells (CD3 + CD8 + ). The experiment was repeated 2 times. Graphs C , D , F-I present the meanL±Ls.d with each symbol represents an individual mouse. Graphs in E present the mean with each symbol represents an individual mouse. Statistical significance was determined using GraphPad Prism by Log-rank (Mantel-Cox) test in B , by Mann-Whitney U test in C , by Repeated measures one-way ANOVA with Tukey’s multiple comparisons test in E , and by unpaired two-sided Welch’s t-test in F-I . *, p < 0.05; **, p < 0.01; ***, p < 0.001, ****, p < 0.0001; “ns” indicates non-statistically significant differences.

Article Snippet: Cells were then stained for 30 minutes at 4 °C with the following anti-mouse antibodies: anti-B220 PerCP-Cy5.5-conjugated (1:100, Biogems, 07131-70-100), anti-IgM BV421-conjugated (1:100, BioLegend, BLG-406506), anti-IgG1 BV421-conjugated (1:100, BioLegend, BLG-406616), anti-GL7 AF647-conjugated (1:100, BioLegend, BLG-144606), anti-FAS (CD95) PE-Vio770-conjugated (1:100, Milteny Biotec, 130-120-291), anti-CD4 BV421-conjugated (1:100, BioLegend, BLG-100438), anti-CD8a FITC-conjugated (1:100, BioLegend, BLG-100706), anti-NK1.1 PE-conjugated (1:100, Milteny Biotec, 130-120-511), anti-CD64 PE-Vio770-conjugated (1:50, Milteny Biotec, 130-119-659), anti-F4/80 APC-conjugated (1:50, Milteny Biotec, 130-116-525), anti-CD69 PE-Vio770-conjugated (1:10, Milteny Biotec, 130-103-977), anti-CD45 Brilliant Violet 711-conjugated (1:100, BioLegend, BLG-103147) antibodies, or anti-human/mouse antibodies: anti-HSP70 Alexa Fluor488-conjugated (1:100, BioLegend, BLG-648004), anti-GRP78 Alexa Fluor488-conjugated (1:100, Santa Cruz Biotechnology, SC-166490), anti-HSC70 Alexa Fluor488-conjugated (1:100, Santa Cruz Biotechnology, SC-7298).

Techniques: Injection, Enzyme-linked Immunosorbent Assay, Flow Cytometry, MANN-WHITNEY

Journal: bioRxiv

Article Title: Decoy Antibodies Block Extracellular HSP70, Prevent Self Signaling and Inhibit Melanoma Cell Survival

doi: 10.64898/2025.12.29.696857

Figure Lengend Snippet: A. Schematic representation of the melanosome-immunization protocol. C57BL/6 mice (n=10 per group) were immunized with 10 µg of melanosomes or PBS, and blood was collected on days 0, 3, 5, 8, 10, 12, and 14 following immunization. Spleens were harvested on day 14 post-immunization. B. Serum IgG response to melanosomes, measured by ELISA, in melanosome-immunized and PBS-immunized mice (n=10 per group). Group mean raw O.D. 650nm values at each time point (days 0, 3, 5, 8, 10, 12, 14); the experiment was repeated 3 times. C. Serum IgG response to melanosomes, measured by ELISA, in individual melanosome-immunized and PBS-immunized mice (n=10 per group) on day 14 post-immunization. The raw O.D. 650 nm values are presented. The four melanosome-immunized mice that were selected for B cell sorting (#18. #30, #31, and #32) are indicated by a red oval; the experiment was repeated 3 times. D. Left: Representative flow cytometry plot and gating strategy of IgG1 + GC B cells from a spleen of a melanosome-immunized mouse. The population that was single-cell sorted is indicated. Gating: lymphocytes → single cells → B cells (B220 + ) → GC B cells (CD95 (FAS) + GL7 + ) → IgG1 + (IgG1 + IgM − ). Right: GC B cells (CD95 (FAS) + GL7+) staining from a spleen of a PBS-immunized mouse. E. Pie charts showing the number of paired IgG1 + Kappa sequences from the 4 melanosome-immunized mice (#18. #30, #31, and #32). The total number of sequences from each mouse is displayed in the center of each pie. Shaded slices indicate expanded B cell clones, while white slices represent unique sequences. F. Binding of mAbs to melanosomes (n = 13, 10 µg/mL), assessed by ELISA (the y axis shows raw O.D. 650 nm values). mGO.53.53 (black line) serves as an isotype control . The three highest binding mAbs Mel321-31, Mel322-34, Mel321-35 are depicted in purple, magenta and red, respectively, while the other 10 mAbs are in gray. G. Binding of mAbs to melanosomes (2.5 µg/mL), as measured by flow cytometry (n=13 mAbs, 10 µg/mL) and detected by staining with anti-mouse IgG conjugated to Alexa Fluor 647. Median fluorescence intensity (MFI, y axis) was calculated for each mAb. The three highest binding mAbs Mel321-31, Mel322-34, Mel321-35 are depicted in purple, magenta and red, respectively, while the other 10 mAbs are in gray. mGO.53 is in black and serves as an isotype control; the experiment was repeated 3 times. H. Quantification of fluorescence intensity of TdT-expressing B16 cells under different treatment conditions: untreated cells (no melanosomes, white circles), cells with 5 µg/mL of external melanosomes (black circles), cells treated with isotype control, mGO.53 (25 µg/mL) plus 5 µg/mL of external melanosomes (gray circles), Mel321-31 (25 µg/mL) plus 5 µg/mL of external melanosomes (purple circles), Mel322-34 (25 µg/mL) plus 5 µg/mL of external melanosomes (pink circles), and Mel321-35 (25 µg/mL) plus 5 µg/mL of external melanosomes (red circles). n=7 per treatment group; the experiment was repeated 3 times. Graphs B-C present the meanL±Ls.d with each symbol represents an individual mouse. Statistical significance was determined using GraphPad Prism by Mann-Whitney U test in B-C and by one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons post-test in H . *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; “ns” indicates non-statistically significant differences.

Article Snippet: Cells were then stained for 30 minutes at 4 °C with the following anti-mouse antibodies: anti-B220 PerCP-Cy5.5-conjugated (1:100, Biogems, 07131-70-100), anti-IgM BV421-conjugated (1:100, BioLegend, BLG-406506), anti-IgG1 BV421-conjugated (1:100, BioLegend, BLG-406616), anti-GL7 AF647-conjugated (1:100, BioLegend, BLG-144606), anti-FAS (CD95) PE-Vio770-conjugated (1:100, Milteny Biotec, 130-120-291), anti-CD4 BV421-conjugated (1:100, BioLegend, BLG-100438), anti-CD8a FITC-conjugated (1:100, BioLegend, BLG-100706), anti-NK1.1 PE-conjugated (1:100, Milteny Biotec, 130-120-511), anti-CD64 PE-Vio770-conjugated (1:50, Milteny Biotec, 130-119-659), anti-F4/80 APC-conjugated (1:50, Milteny Biotec, 130-116-525), anti-CD69 PE-Vio770-conjugated (1:10, Milteny Biotec, 130-103-977), anti-CD45 Brilliant Violet 711-conjugated (1:100, BioLegend, BLG-103147) antibodies, or anti-human/mouse antibodies: anti-HSP70 Alexa Fluor488-conjugated (1:100, BioLegend, BLG-648004), anti-GRP78 Alexa Fluor488-conjugated (1:100, Santa Cruz Biotechnology, SC-166490), anti-HSC70 Alexa Fluor488-conjugated (1:100, Santa Cruz Biotechnology, SC-7298).

Techniques: Enzyme-linked Immunosorbent Assay, FACS, Flow Cytometry, Staining, Clone Assay, Binding Assay, Control, Fluorescence, Expressing, MANN-WHITNEY