cd5 microbeads positive selection  (Miltenyi Biotec)

Journal: Cancer immunology research

Article Title: Intermittent MEK inhibition with GITR co-stimulation rescues T-cell function for increased efficacy with CTLA-4 blockade in solid tumor models

doi: 10.1158/2326-6066.CIR-23-0729

Figure Lengend Snippet: ( A-D) CD5+ T cells purified from naïve C57BL/6 spleens were labelled with CellTrace Violet (CTV), sub-optimally stimulated with α-CD3/α-CD28 Dynabeads, and treated with DMSO, continuous CKI27 (72hr), or washout CKI27 (16hr on, 52hr off); n=3. Experiments were repeated 2–3 times. (A) Proliferation as measured by % of CTV low CD8+ and CD4+ T cells. (B) Proliferation fold change of CD8+ and CD4+ T cells was calculated by normalizing to DMSO. (C) FACS analysis of co-inhibitory, co-stimulatory, and activation markers on CD8+ and CD4+ T cells. Heatmaps represent fold changes of positive percentages of each marker normalized to DMSO. (D) Cytokine analysis of supernatants collected from CD5+ T cells. Heatmap represents fold changes of concentrations (pg/mL) of proteins normalized to DMSO. (E) DMSO, continuous CKI27, or washout CKI27 treated OT-1 primed T cells were co-cultured with SIINFEKL peptide pulsed B16-YFP. Killing was assessed after 48hr using a Celigo Imaging Cytometer; n=5–6. Data are shown as mean±SEM. One-way ANOVA test with Tukey’s correction for multiple comparisons was used. Significance levels are indicated by asterisks (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001)

Article Snippet: For mouse T cell–proliferation assays: T cells isolated from the spleens of naïve C57BL/6 mice (JAX, #00664) were purified with the EasySep Mouse T cell Isolation Kit (Stemcell, #19851) and CD5 + T cells were purified with CD5 + Microbeads positive selection (Miltenyi, #130-049-301).

Techniques: Inhibition, Purification, Activation Assay, Marker, Cell Culture, Imaging, Cytometry

Journal: Cancer immunology research

Article Title: Intermittent MEK inhibition with GITR co-stimulation rescues T-cell function for increased efficacy with CTLA-4 blockade in solid tumor models

doi: 10.1158/2326-6066.CIR-23-0729

Figure Lengend Snippet: (A-C) CD5 + T cells purified from naïve C57BL/6 spleens were labelled with CTV, sub-optimally stimulated with anti-CD3/anti-CD28 Dynabeads, and treated with DMSO, continuous CKI27 (72hr), washout CKI27 (16hr on, 52hr off), isotypes, anti-GITR, and/or anti-OX-40; n=3. Experiments were repeated 2–3 times. (A) Proliferation as measured by % of CTV low CD8+ and CD4+ T cells. Data are shown as mean±SEM. Two-way ANOVA test with Bonferroni’s correction for multiple comparisons was used. Significance levels are indicated by asterisks (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001) (B) FACS analysis of co-inhibitory, co-stimulatory, and activation markers on CD8 + and CD4 + T cells. Heatmaps represent fold changes of positive percentages of each marker normalized to DMSO. (C) Cytokine analysis of supernatants collected from CD5+ T cells. Heatmap represents fold changes of concentrations (pg/mL) of proteins normalized to DMSO. (D) DMSO, washout CKI27, isotypes, anti-GITR, and/or anti-OX-40 treated OT-1 primed T cells were co-cultured with unpulsed or SIINFEKL peptide pulsed B16-YFP. Killing was assessed after 48hr using a Celigo Imaging Cytometer; n=4. Data are shown as mean±SEM. One-way ANOVA test with Tukey’s correction for multiple comparisons was used. Significance levels are indicated by asterisks (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001) (E) Sub-optimally anti-CD3/anti-CD28 stimulated CD8 + T cells were treated with DMSO, CKI27, isotypes, anti-GITR, and/or anti-OX-40 for 72hr. Lysates were assayed by immunoblotting to determine the level of ERK, MEK, p38, JNK, p70S6k, and p65 phosphorylation. (F) Quantitation of the relative density of bands were normalized to the loading control (vinculin). The ratio was calculated by normalizing phosphorylated protein to total protein.

Article Snippet: For mouse T cell–proliferation assays: T cells isolated from the spleens of naïve C57BL/6 mice (JAX, #00664) were purified with the EasySep Mouse T cell Isolation Kit (Stemcell, #19851) and CD5 + T cells were purified with CD5 + Microbeads positive selection (Miltenyi, #130-049-301).

Techniques: Cell Function Assay, Protein-Protein interactions, Purification, Activation Assay, Marker, Cell Culture, Imaging, Cytometry, Western Blot, Phospho-proteomics, Quantitation Assay, Control

Journal: Frontiers in Veterinary Science

Article Title: Flow cytometric-based detection of CD80 is a useful diagnostic marker of acute myeloid leukemia in dogs

doi: 10.3389/fvets.2024.1405297

Figure Lengend Snippet: Conjugated antibodies used for triple-labeling cells in acute leukemia immunophenotyping panels after April 2021, including their target antigen and registry number (when available) or source.

Article Snippet: B cells, T cells, and monocytes were then individually isolated from PBMC using conjugated antibodies against CD21, CD5, and CD14, followed by anti-murine IgG magnetic microbeads for CD14 and CD21 (Miltenyi Biotec Cat# 130–048-402, RRID:AB_244361) and anti-rat IgG magnetic microbeads for CD5 (Miltenyi Biotec Cat# 130–048-502, RRID:AB_244364) and a magnetic column (LS column, Miltenyi Biotec, Gaithersburg, MD, United States) as we described previously for isolating CD14 + monocytes ( ).

Techniques:

Journal: Frontiers in Veterinary Science

Article Title: Flow cytometric-based detection of CD80 is a useful diagnostic marker of acute myeloid leukemia in dogs

doi: 10.3389/fvets.2024.1405297

Figure Lengend Snippet: Conjugated antibodies used for triple-labeling cells in a lymphoma immunophenotyping panel used for routine diagnostic testing after April 2021, including their target antigen*.

Article Snippet: B cells, T cells, and monocytes were then individually isolated from PBMC using conjugated antibodies against CD21, CD5, and CD14, followed by anti-murine IgG magnetic microbeads for CD14 and CD21 (Miltenyi Biotec Cat# 130–048-402, RRID:AB_244361) and anti-rat IgG magnetic microbeads for CD5 (Miltenyi Biotec Cat# 130–048-502, RRID:AB_244364) and a magnetic column (LS column, Miltenyi Biotec, Gaithersburg, MD, United States) as we described previously for isolating CD14 + monocytes ( ).

Techniques: Diagnostic Assay

Journal: Frontiers in Veterinary Science

Article Title: Flow cytometric-based detection of CD80 is a useful diagnostic marker of acute myeloid leukemia in dogs

doi: 10.3389/fvets.2024.1405297

Figure Lengend Snippet: Flow cytometric dot plots of anti-CD80 antibody labeling of leukocytes in blood from healthy dogs. (A) Three different cell populations were identified on a forward and side scatter plot, corresponding to neutrophils, monocytes, and lymphocytes. The cells were double-labeled with anti-CD80-APC and -CD14-PE antibodies, with CD14 being used as a monocyte marker. CD80 was only expressed on CD14 + monocytes and CD14 − neutrophils but not lymphocytes (CD80 − /CD14 − ). The few CD14 + and CD14 − cells in the neutrophil and monocyte gates likely represent low numbers of monocytes and neutrophils in the respective gates. The CD80 − /CD14 − cells in neutrophil and monocyte gates could be large lymphocytes (representative result from 1 of 4 dogs). (B) Triple-labeling of dog leukocytes with CD80-APC, CD21-PE and CD5-FITC shows that CD21 + B cells and CD5 + T cells are CD80 − (representative result from 1 of 3 dogs). All leukocyte events were combined for analysis vs. splitting the events into different gates based on forward and side scatter.

Article Snippet: B cells, T cells, and monocytes were then individually isolated from PBMC using conjugated antibodies against CD21, CD5, and CD14, followed by anti-murine IgG magnetic microbeads for CD14 and CD21 (Miltenyi Biotec Cat# 130–048-402, RRID:AB_244361) and anti-rat IgG magnetic microbeads for CD5 (Miltenyi Biotec Cat# 130–048-502, RRID:AB_244364) and a magnetic column (LS column, Miltenyi Biotec, Gaithersburg, MD, United States) as we described previously for isolating CD14 + monocytes ( ).

Techniques: Antibody Labeling, Labeling, Marker

Journal: Frontiers in Veterinary Science

Article Title: Flow cytometric-based detection of CD80 is a useful diagnostic marker of acute myeloid leukemia in dogs

doi: 10.3389/fvets.2024.1405297

Figure Lengend Snippet: Labeling of monocytes, B cells, T cells, and neutrophils isolated from the blood of healthy dogs (representative results from 1 of 2–3 dogs for each cell type) with the anti-CD80 antibody. (A-C) Monocytes, B cells, and T cells were isolated from peripheral blood mononuclear cells (PBMCs) using immunomagnetic beads and anti-CD14-PE, -CD21-FITC, and -CD5-FITC antibodies, respectively. The first panel shows the forward and side scatter events in PBMCs while the second panel shows the forward and side scatter events of isolated cells. The third panel is a fluorescent quadrant plot of double-labeled cells after adding the anti-CD80-APC antibody. The fourth panel shows a representative modified Wright’s-stained image of a cytospin smear of the isolated cells on which 100-cell differential cell counts were done (scale bar = 20 μm). (A) CD14-PE-isolated cells were mostly CD80 + monocytes (84% of a differential cell count), with a few contaminating neutrophils (10%) that were weakly CD14 + (arrows, third and fourth panels). A few monocytes had cytoplasmic vacuoles (fourth panel). Lymphocytes were negative for CD80 − /CD14 − (lower left quadrant, third panel, 6%). (B) CD21-FITC-isolated cells were mostly lymphocytes, which were CD80 − (third panel). Lymphocytes were primarily small cells, some of which had clefted or convoluted nuclei (variants of normal), with a few small or large reactive forms (fourth panel). (C) CD5-FITC-isolated cells were mostly lymphocytes, which were CD80 − (third panel). Lymphocytes were small cells with a few large or reactive forms. Several lymphocytes had a few clear cytoplasmic vacuoles, which could be due to the isolation procedure (fourth panel). (D) Neutrophils were isolated from the 1.077/1.119 interface of the double-density gradient used to obtain PBMCs and were single-labeled with the anti-CD80-APC antibody. The first panel shows a forward vs. side scatter plot of the isolated neutrophils and the second panel is a CD80 fluorescence vs. side scatter dot plot (blue) with overlaid hamster-APC isotype (red), showing neutrophils are CD80 + . The third panel shows a representative modified Wright’s-stained image of a cytospin smear of the isolated cells, which were primarily segmented neutrophils. The vacuolated cytoplasm in one neutrophil is likely an artifact of the isolation procedure (scale bar = 20 μm).

Article Snippet: B cells, T cells, and monocytes were then individually isolated from PBMC using conjugated antibodies against CD21, CD5, and CD14, followed by anti-murine IgG magnetic microbeads for CD14 and CD21 (Miltenyi Biotec Cat# 130–048-402, RRID:AB_244361) and anti-rat IgG magnetic microbeads for CD5 (Miltenyi Biotec Cat# 130–048-502, RRID:AB_244364) and a magnetic column (LS column, Miltenyi Biotec, Gaithersburg, MD, United States) as we described previously for isolating CD14 + monocytes ( ).

Techniques: Labeling, Isolation, Modification, Staining, Cell Counting, Fluorescence

Journal: Frontiers in Veterinary Science

Article Title: Flow cytometric-based detection of CD80 is a useful diagnostic marker of acute myeloid leukemia in dogs

doi: 10.3389/fvets.2024.1405297

Figure Lengend Snippet: Percentage differential cell counts (mean and range) from modified Wright’s-stained cytospin smears of isolated monocytes, B cells, T cells, and neutrophils.

Article Snippet: B cells, T cells, and monocytes were then individually isolated from PBMC using conjugated antibodies against CD21, CD5, and CD14, followed by anti-murine IgG magnetic microbeads for CD14 and CD21 (Miltenyi Biotec Cat# 130–048-402, RRID:AB_244361) and anti-rat IgG magnetic microbeads for CD5 (Miltenyi Biotec Cat# 130–048-502, RRID:AB_244364) and a magnetic column (LS column, Miltenyi Biotec, Gaithersburg, MD, United States) as we described previously for isolating CD14 + monocytes ( ).

Techniques: Modification, Isolation

Journal: Frontiers in Veterinary Science

Article Title: Flow cytometric-based detection of CD80 is a useful diagnostic marker of acute myeloid leukemia in dogs

doi: 10.3389/fvets.2024.1405297

Figure Lengend Snippet: Positive labeling with the anti-CD80 antibody in tumor cells in dogs with hematopoietic neoplasms.

Article Snippet: B cells, T cells, and monocytes were then individually isolated from PBMC using conjugated antibodies against CD21, CD5, and CD14, followed by anti-murine IgG magnetic microbeads for CD14 and CD21 (Miltenyi Biotec Cat# 130–048-402, RRID:AB_244361) and anti-rat IgG magnetic microbeads for CD5 (Miltenyi Biotec Cat# 130–048-502, RRID:AB_244364) and a magnetic column (LS column, Miltenyi Biotec, Gaithersburg, MD, United States) as we described previously for isolating CD14 + monocytes ( ).

Techniques: Labeling

Journal: Immunity

Article Title: Ambient oxygen levels regulate intestinal dysbiosis and GVHD severity after allogeneic stem cell transplantation

doi: 10.1016/j.immuni.2023.01.007

Figure Lengend Snippet: Lethally irradiated (1000cGy) 129 mice received bone marrow (5x106 cells) and purified CD5+T cells (2x106 cells) from C57BL/6J donors in different institution (Memorial Sloan Kettering Cancer Center). (A) Tissue hypoxia was determined with Pimonidazole (left, scale bar= 50μm). Quantification of intestinal O2 levels in intestine from recipients (right). Samples were collected 4, 8, 14, and 21days after BMT (Naïve: N=3, BM only: N=4, BM+T: day4, 8, 14 N=4, day21 N=3). (B) Stool microbial composition was determined by 16S rRNA gene sequencing. (C) The classification of obligate/facultative (O/F) anaerobe ratio was determined. Two-tailed unpaired t-test (A) was used to determine significance (mean ± s.e.m.). **P < 0.01.

Article Snippet: CD5 (Ly-1) MicroBeads, mouse , Miltenyi Biotec , Cat# 130-049-301.

Techniques: Irradiation, Purification, Sequencing, Two Tailed Test

Journal: Immunity

Article Title: Ambient oxygen levels regulate intestinal dysbiosis and GVHD severity after allogeneic stem cell transplantation

doi: 10.1016/j.immuni.2023.01.007

Figure Lengend Snippet: Key resources table

Article Snippet: CD5 (Ly-1) MicroBeads, mouse , Miltenyi Biotec , Cat# 130-049-301.

Techniques: Polymer, Recombinant, Lysis, Extraction, Western Blot, Plasmid Preparation, Picogreen Assay, Membrane, Reverse Transcription, Iron Assay, Sequencing, Software