Journal: Current protocols in cytometry

Article Title: Monitoring of Measurable Residual Disease in Multiple Myeloma by Multiparametric Flow Cytometry

doi: 10.1002/cpcy.63

Figure Lengend Snippet: All plots shown are gated on [R1 & R2 & R3 & NOT (R4 & R5 &R6)]. (A) Mast cells (defined as CD27-, CD117br) are identified by drawing an elliptical region (R8) on a bivariate plot of CD27 BV510 vs. CD117 APC. (B) An elliptical region (R9) is placed on a bivariate plot of SSC-A vs. CD45 PCPCy5.5 to identify erythroid precursors (defined as CD45-, SSClo). (C & D) B cells (defined as CD56-, CD19+) are identified by placing a rectangular region (R10) on a bivariate plot of CD56 PE vs. CD19 PECy7. B cell are then delineated using a bivariate plot of CD45 PCPCy5.5 vs. CD81 APCH7. A region (R11) is then drawn to identify hematogones (defined as CD45dim, CD81br).

Article Snippet: Use the gating strategies detailed in Step 3 – 12 below to create a series of bivariate plots for identifying appropriate negative and positive populations used for calculating stain indices for each mAb. list-behavior=unordered prefix-word= mark-type=disc max-label-size=0 Stain index is a well-accepted and reliable measure that can be used to evaluate the performance of individual mAb employed in a multi-color tube; it can be calculated using the following formula: S t a i n I n d e x = M e d i a n F l u o r e s c e n c e I n t e n s i t i e s o f ( P o s i t i v e P o p u l a t i o n − N e g a t i v e P o p u l a t i o n ) 2 x R o b u s t S t a n d a r d D e v i a t i o n o f N e g a t i v e P o p u l a t i o n A summary of the gating definitions used to determine negative and positive populations for analysis of this equation in a one parameter histogram can be found in and the minimum recommended Stain Index for each fluorochrome-conjugated mAb can be found in . table ft1 table-wrap mode="anchored" t5 Table 3. caption a7 Tested Markers Negative Population Positive Population Phenotype Generic Name Phenotype Generic Name CD45 CD45-, SSClo Erythroid Precursors CD19-, CD56-, CD45br, SSClo T Cells CD19 CD45br, CD56+, SSClo NK Cells CD19+, CD45br, SSClo B Cells CD27 CD45+, CD117br Mast Cells CD19-, CD56-, CD45br, SSClo T Cells CD81 CD45dim,SSChi Granulocytes CD19-, CD56-, CD45br, SSClo T Cells CD56 CD19+, CD56-, CD45br, SSClo B Cells CD19-, CD56+, CD45br, SSClo NK Cells CD117 CD19-, CD56-, CD45br, SSClo T Cells CD27, CD117br Mast Cells CD138 CD19+, CD56-, CD45br, SSClo B Cells CD45dim, CD81+ Spiked Control a CD38 CD19+, CD81dim Mature B CD19+, CD81+ B-progenitors cKappa CD19+, CD45br, cLambda+, SSClo cLambda+ B Cells CD19+, CD45br, cLambda-, SSClo cLambda- B Cells cLambda CD19+, CD45br, cKappa+, SSClo cKappa+ B cells CD19+, CD45br, cKappa-, SSClo cKappa- B cells Open in a separate window a A procedural control for immunophenotyping, CD-Chex CD103 TM Plus (Streck, Omaha, NE), which also expresses CD138 is spiked into the bone marrow sample and used as the positive population.

Techniques:

Journal: Current protocols in cytometry

Article Title: Monitoring of Measurable Residual Disease in Multiple Myeloma by Multiparametric Flow Cytometry

doi: 10.1002/cpcy.63

Figure Lengend Snippet: Immunophenotypic definition of the negative and positive populations used for calculating Stain Index.

Article Snippet: Use the gating strategies detailed in Step 3 – 12 below to create a series of bivariate plots for identifying appropriate negative and positive populations used for calculating stain indices for each mAb. list-behavior=unordered prefix-word= mark-type=disc max-label-size=0 Stain index is a well-accepted and reliable measure that can be used to evaluate the performance of individual mAb employed in a multi-color tube; it can be calculated using the following formula: S t a i n I n d e x = M e d i a n F l u o r e s c e n c e I n t e n s i t i e s o f ( P o s i t i v e P o p u l a t i o n − N e g a t i v e P o p u l a t i o n ) 2 x R o b u s t S t a n d a r d D e v i a t i o n o f N e g a t i v e P o p u l a t i o n A summary of the gating definitions used to determine negative and positive populations for analysis of this equation in a one parameter histogram can be found in and the minimum recommended Stain Index for each fluorochrome-conjugated mAb can be found in . table ft1 table-wrap mode="anchored" t5 Table 3. caption a7 Tested Markers Negative Population Positive Population Phenotype Generic Name Phenotype Generic Name CD45 CD45-, SSClo Erythroid Precursors CD19-, CD56-, CD45br, SSClo T Cells CD19 CD45br, CD56+, SSClo NK Cells CD19+, CD45br, SSClo B Cells CD27 CD45+, CD117br Mast Cells CD19-, CD56-, CD45br, SSClo T Cells CD81 CD45dim,SSChi Granulocytes CD19-, CD56-, CD45br, SSClo T Cells CD56 CD19+, CD56-, CD45br, SSClo B Cells CD19-, CD56+, CD45br, SSClo NK Cells CD117 CD19-, CD56-, CD45br, SSClo T Cells CD27, CD117br Mast Cells CD138 CD19+, CD56-, CD45br, SSClo B Cells CD45dim, CD81+ Spiked Control a CD38 CD19+, CD81dim Mature B CD19+, CD81+ B-progenitors cKappa CD19+, CD45br, cLambda+, SSClo cLambda+ B Cells CD19+, CD45br, cLambda-, SSClo cLambda- B Cells cLambda CD19+, CD45br, cKappa+, SSClo cKappa+ B cells CD19+, CD45br, cKappa-, SSClo cKappa- B cells Open in a separate window a A procedural control for immunophenotyping, CD-Chex CD103 TM Plus (Streck, Omaha, NE), which also expresses CD138 is spiked into the bone marrow sample and used as the positive population.

Techniques: Staining, Control

Journal: Current protocols in cytometry

Article Title: Monitoring of Measurable Residual Disease in Multiple Myeloma by Multiparametric Flow Cytometry

doi: 10.1002/cpcy.63

Figure Lengend Snippet: The strategy used to assess the performance of each mAb in the panel relies on defining negative (blue events) and positive (red events) populations for calculating stain indices. This is accomplished by first subtracting the median intensity of negative population from the median intensity of the positive population and dividing the number by 2 times the robust standard deviation of the negative population. A list of gating definitions can be found in Table 2. (A) For CD45, erythroid precursors are used as the negative population defined as SSClo, CD45- events (r1; blue dots) and T cells as the positive population defined as CD19-, CD56-, CD45br, SSClo events (r2 & r3; red dots). (B) For CD19, NK cells are used as the negative population defined as CD45br, CD56+ events (r4: blue dots) and B cells as the positive population defined as CD19+, CD45br events (r5; red dots). (C) For CD27, mast cells are used as the negative population defined as CD45dim, CD117br events (r6; blue dots) and T cells as the positive population defined as CD19-, CD56-, CD45br, SSClo events (r2 & r7; red dots). (D) For CD81, granulocytes are used as the negative population defined as CD45dim, SSChi events (r8; blue dots) and T cells as the positive population defined as CD19-, CD56-, CD45br, SSClo events (r9 & r10; red dots). (E) For CD56, B cells are used as the negative population defined as CD19+, CD56-, CD45br, SSClo events (r2 & r11; blue dots) and NK cells as the positive population defined as CD19-, CD56+, CD45br, SSClo events (r2 & r12; red dots). (F) For CD117, T cells are used as the negative population defined as CD19-, CD56-, CD45br, SSClo events (r2 & r13; blue dots) and mast cells as the positive population defined as CD27-, CD117br events (r14; red dots). (G) For CD138, B cells are used as the negative population defined as CD19+, CD56-, SSClo events (r2 & r15; blue dots) and CD-Chex CD103™ Plus cells as the positive population defined as CD81+, CD45dim events (r16; red dots). (H) For CD38, mature B cells are used as the negative population defined as CD19+, CD81dim events (r17; blue dots) and B progenitors as the positive population defined as CD19+, CD81br events (r18; red dots). (I) For cKappa, cLambda+ B cells are used as the negative population defined as CD19+, cLambda+, CD45br, SSClo events (r2 & r19; blue dots) and cLambda- B cells as the positive population defined as CD19+, cLambda-, CD45br, SSClo (r2 & r20; red dots). (J) For cLambda, cKappa+ B cells are used as the negative population defined as CD19+, cKappa+, CD45br, SSClo events (r2 & r21; blue dots) and cKappa- B cells as the positive population defined as CD19+, cKappa-, CD45br, SSClo (r2 & r22; red dots).

Article Snippet: Use the gating strategies detailed in Step 3 – 12 below to create a series of bivariate plots for identifying appropriate negative and positive populations used for calculating stain indices for each mAb. list-behavior=unordered prefix-word= mark-type=disc max-label-size=0 Stain index is a well-accepted and reliable measure that can be used to evaluate the performance of individual mAb employed in a multi-color tube; it can be calculated using the following formula: S t a i n I n d e x = M e d i a n F l u o r e s c e n c e I n t e n s i t i e s o f ( P o s i t i v e P o p u l a t i o n − N e g a t i v e P o p u l a t i o n ) 2 x R o b u s t S t a n d a r d D e v i a t i o n o f N e g a t i v e P o p u l a t i o n A summary of the gating definitions used to determine negative and positive populations for analysis of this equation in a one parameter histogram can be found in and the minimum recommended Stain Index for each fluorochrome-conjugated mAb can be found in . table ft1 table-wrap mode="anchored" t5 Table 3. caption a7 Tested Markers Negative Population Positive Population Phenotype Generic Name Phenotype Generic Name CD45 CD45-, SSClo Erythroid Precursors CD19-, CD56-, CD45br, SSClo T Cells CD19 CD45br, CD56+, SSClo NK Cells CD19+, CD45br, SSClo B Cells CD27 CD45+, CD117br Mast Cells CD19-, CD56-, CD45br, SSClo T Cells CD81 CD45dim,SSChi Granulocytes CD19-, CD56-, CD45br, SSClo T Cells CD56 CD19+, CD56-, CD45br, SSClo B Cells CD19-, CD56+, CD45br, SSClo NK Cells CD117 CD19-, CD56-, CD45br, SSClo T Cells CD27, CD117br Mast Cells CD138 CD19+, CD56-, CD45br, SSClo B Cells CD45dim, CD81+ Spiked Control a CD38 CD19+, CD81dim Mature B CD19+, CD81+ B-progenitors cKappa CD19+, CD45br, cLambda+, SSClo cLambda+ B Cells CD19+, CD45br, cLambda-, SSClo cLambda- B Cells cLambda CD19+, CD45br, cKappa+, SSClo cKappa+ B cells CD19+, CD45br, cKappa-, SSClo cKappa- B cells Open in a separate window a A procedural control for immunophenotyping, CD-Chex CD103 TM Plus (Streck, Omaha, NE), which also expresses CD138 is spiked into the bone marrow sample and used as the positive population.

Techniques: Staining, Standard Deviation

Journal: Cancer immunology, immunotherapy : CII

Article Title: Tissue-resident memory CD103+CD8+ T cells in colorectal cancer: its implication as a prognostic and predictive liver metastasis biomarker.

doi: 10.1007/s00262-024-03709-2

Figure Lengend Snippet: Fig. 3 The expression of CD31 and α-SMA in clinical samples and the anti-angiogenic therapy promotes infiltration of CD103+CD8+ TRMs in tumor. A The expression of CD31 and α-SMA in CRC and CRC liver metastasis tissue. B The infiltra- tion level of CD103+CD8+ TRMs in the α-SMA high and low groups. C Comparison of median number and interquar- tile range of CD103+CD8+ TRMs. D The relationship of CD103+CD8+ TRMs and α-SMA+ vessel. E–F The infil- tration level of CD103+CD8+ TRMs in CRC patients who responded and non-respond to bevacizumab treatment. G Average tumor growth curves showing tumor volume in mice treated with either control or the Bevacizumab (n = 5 mice per group). H–K Representative flow cytometry plots (H) and the percentage of CD103+CD8+ TRMs (I) and representative flow cytometry plots (J) and the percentage of IFN-γ+ CD103+CD8+ TRMs (K) isolated from mice on day 21 (n = 5 mice per group). (Immunofluorescent staining, × 400, The white triangle in the tissue is CD103+CD8+ TRMs). CRC, colorectal cancer; TRM, tissue-resident memory T cell. α-SMA, alpha-smooth muscle actin. *p < 0.05; **p < 0.01; ***p < 0.001

Article Snippet: And 1 μl of fluorescein isothiocyanate (FITC) anti-mouse CD8 (cat. 11-0081-81, eBioScience), phycoerythrin (PE) anti-mouse CD103 (cat. E-AB-F1090D, Elabscience), and APC anti-mouse IFN-γ (cat. E-AB-F1101UE, Elabscience) were added to the tube, respectively.

Techniques: Expressing, Comparison, Control, Flow Cytometry, Isolation, Staining

Journal: Cell death discovery

Article Title: Concerted BAG3 and SIRPα blockade impairs pancreatic tumor growth.

doi: 10.1038/s41420-022-00817-9

Figure Lengend Snippet: Fig. 4 CD11c+ dendritic cells in mice tumors after antibodies treatments. A CD11c+ dendritic cells in excised tumors were revealed by confocal immunofluorescence microscopy with an anti-CD11c monoclonal antibody. Nuclei were counterstained with DAPI. Three to five fields, according to their size, of four tumors per group were examined. B Representative images of CD11c positivity. Images were acquired in sequential scan mode using the same acquisitions parameters (laser intensities, gain photomultipliers, pinhole aperture, objective×40, zoom 1) to compare treated samples and controls. Non-parametric Kruskal–Wallis test with Dunn’s correction was used for data analysis. C Expression of CD103 antigen (red) in CD11c (green)-positive cells.

Article Snippet: Sections were then incubated with anti-CD8 monoclonal antibody (C8/144B, Thermo Fisher 1:25), anti-CD11c monoclonal antibody (ab33483, Abcam, at 1:25), anti-CD103 monoclonal antibody (DM3536P, OriGene Technologies, at 1:25), anti-α-SMA antibody (A2547, SigmaAldrich, at 1:350) overnight at 4 °C in a humidified chamber.

Techniques: Microscopy, Expressing

Journal: Cell reports

Article Title: Single-cell RNA sequencing identifies a population of human liver-type ILC1s

doi: 10.1016/j.celrep.2022.111937

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: CD103 AF700 (Ber-ACT8) , NOVUS , RRID: AB_11189940; Cat# NBP1-97564AF700.

Techniques: Blocking Assay, Recombinant, Clinical Proteomics, Nucleic Acid Electrophoresis, Software, Inverted Microscopy

Journal: bioRxiv

Article Title: HIV-specific CD8 + T-cells in tonsils express exhaustive T RM -like signatures

doi: 10.1101/2021.11.04.467061

Figure Lengend Snippet: A . Blood and tonsil mononuclear cells pregated on CD8 T-cells by flow cytometry for perforin and granzyme B. B . Expression intensity of CD69, CD103, CXCR5, CD127, PD-1, Perforin and granzyme B in multidimensional tSNE space for CD8 + T-cells, gated from total CD45 + T-cells. Peripheral blood (blue), tonsil tissue (orange). C . Percentage of blood and tonsil CD8 + T-cells expressing indicated markers. D . Schematic of protocol for isolate of HIV + CD8 + T cells from peripheral blood and tonsil tissue by scRNA-seq using Seq-Well v3 (top). UMAP of CD8 + T cells coloured by tissue source (bottom). E . Heatmap of z-scored gene expression of CD8 + T cells illustrating differentially expressed genes between blood and tonsil. Selective specifically expressed genes are marked alongside. F . Violin plots showing selected genes for cytolytic, transcription factors, tissue-resident memory and immune suppressive markers, expressed in CD8 + T cells. FDR-adjusted p<0.05; full results can be found in Supplementary Table S1. G . Fluorescent immunohistochemistry of whole tonsil section (left) from HIV uninfected donor shown by CD8, CD4 and merged panels (middle) with quantification using 10 unrelated areas identified outside follicles (outside GCs) and within follicular germinal centers ( inside GCs). P-values by Kruskal-Wallis multi comparisons.

Article Snippet: Exactly 4μm sections were cut, deparaffinized and stained with the following unlabelled primary antibodies: CD8 (clone: C8/144B, Dako), CD4 (clone: 4B12, Dako), p24 (clone: Kal-1, Dako), CXCR5 (clone: MU5UBEE, Thermofisher Scientific), Granzyme B (clone: 23 H8L20, Thermofisher Scientific), CD103 (clone: NBP1-88142, Novus Biologicals) and CD69 (clone: 15B5G2, Novus Biologicals).

Techniques: Flow Cytometry, Expressing, Gene Expression, Immunohistochemistry

Journal: bioRxiv

Article Title: HIV-specific CD8 + T-cells in tonsils express exhaustive T RM -like signatures

doi: 10.1101/2021.11.04.467061

Figure Lengend Snippet: A . Percentage of blood (circles) and tonsil (triangles) CD8 + T cells expressing CD69, CD103, CXCR5, CD27, CD127, PD-1, granzyme B and perforin markers between HIV − and HIV + . Red data point indicates viremic (detectable HIV RNA) and red/black data point indicates suppressed (undetectable HIV RNA). P-values calculated using ordinary one-way ANOVA with horizontal bars representing median values with the level of significance indicated with p-value. B . Representative FACS plot of CD8 + T-cells from HIV-(grey triangles) and HIV + (red triangles includes both ART suppressed and viremic individuals) tonsils for co-expression of CD69/CD103 and PD-1/CD127 with cumulative data shown with horizontal bars representing median values and p-values by Kruskal-wallis multiple comparisons. C . CD8, CD69, CD103 fluorescent immunohistochemistry of whole tonsil sections zoomed in at individual GCs for three independent donors from HIV − (left), HIV + ART + (middle) and HIV + ART − (right) with individuals markers shown. D . Same as in C but for CD8, HIV-p24 and PD-1.

Article Snippet: Exactly 4μm sections were cut, deparaffinized and stained with the following unlabelled primary antibodies: CD8 (clone: C8/144B, Dako), CD4 (clone: 4B12, Dako), p24 (clone: Kal-1, Dako), CXCR5 (clone: MU5UBEE, Thermofisher Scientific), Granzyme B (clone: 23 H8L20, Thermofisher Scientific), CD103 (clone: NBP1-88142, Novus Biologicals) and CD69 (clone: 15B5G2, Novus Biologicals).

Techniques: Expressing, Immunohistochemistry

Journal: bioRxiv

Article Title: HIV-specific CD8 + T-cells in tonsils express exhaustive T RM -like signatures

doi: 10.1101/2021.11.04.467061

Figure Lengend Snippet: A . Representative FACS plot of the gating strategy of CD8 + T-cells from tonsil cells to detect naïve, T N (CCR7 + CD45RA + ), central memory (T CM , CCR7 + CD45RA − ), effector memory (T EM , CCR7 − CD45RA − ) and T EMRA (CCR7 − CD45RA + ). B . Different memory subset distribution within blood and tonsil CD8 + T-cells for central memory, T CM (blue), Effector memory, T EM (red), transitional, T EMRA (orange), T Naive (grey) with cumulative memory subset distribution of CD8 + T cells for blood (circles, left) and tonsil (triangles, right). C . Distribution of blood central memory (T CM ), transitional memory (T EMRA ), effector memory (T EM ) and naïve subsets within CD127, CD69, PD-1, perforin and granzyme B expressing CD8 + T-cells cumulative for all study participants in HIV − (grey) and HIV + (red). D . Same as in C but showing data from tonsil CD8 + T-cells. P-values calculated using ordinary one-way ANOVA with horizontal bars representing median values with the level of significance indicated above. E . The frequency of CD103, CD69, CD127, perforin, and granzyme B (Granz B) cells measured on PD-1 + (left) and PD-1 − (right) CD8 + T-cells from blood in HIV + (red) and HIV − (grey) individuals. F . Same as in E but showing data from tonsil CD8 + T-cells. P-values calculated using Paired Student’s t test. Horizontal bars represent median values.

Article Snippet: Exactly 4μm sections were cut, deparaffinized and stained with the following unlabelled primary antibodies: CD8 (clone: C8/144B, Dako), CD4 (clone: 4B12, Dako), p24 (clone: Kal-1, Dako), CXCR5 (clone: MU5UBEE, Thermofisher Scientific), Granzyme B (clone: 23 H8L20, Thermofisher Scientific), CD103 (clone: NBP1-88142, Novus Biologicals) and CD69 (clone: 15B5G2, Novus Biologicals).

Techniques: Expressing

Journal: bioRxiv

Article Title: HIV-specific CD8 + T-cells in tonsils express exhaustive T RM -like signatures

doi: 10.1101/2021.11.04.467061

Figure Lengend Snippet: A . Representative flow plots showing CMV-(top) and HIV-specific (bottom) tetramer stains of blood (left) and tonsil tissue (right) from the same participant. B . Heat map showing expression frequencies for the indicated markers among CD8 + T cells from CMV tetramer and HIV tetramer specific CD8 + T-cells in blood (top) and tonsil (bottom) gated CD8 + T-cells with frequencies for each tetramer population indicated in the bar below from blue (0%) to red (100%). C . Frequencies of CD69, CD103, PD-1 and CD127 from HIV-, CMV-, and non-specific (‘CD8’) CD8 + T-cells within blood (left) and tonsil (right) tissue. P-values calculated using ordinary one-way ANOVA with horizontal bars representing median values with the level of significance indicated above.

Article Snippet: Exactly 4μm sections were cut, deparaffinized and stained with the following unlabelled primary antibodies: CD8 (clone: C8/144B, Dako), CD4 (clone: 4B12, Dako), p24 (clone: Kal-1, Dako), CXCR5 (clone: MU5UBEE, Thermofisher Scientific), Granzyme B (clone: 23 H8L20, Thermofisher Scientific), CD103 (clone: NBP1-88142, Novus Biologicals) and CD69 (clone: 15B5G2, Novus Biologicals).

Techniques: Expressing

Journal: bioRxiv

Article Title: HIV-specific CD8 + T-cells in tonsils express exhaustive T RM -like signatures

doi: 10.1101/2021.11.04.467061

Figure Lengend Snippet: A . Workflow of single-cell RNA sequencing from HIV infected tonsil tissue isolated CD8 + T-cells pre-sorted on HIV-, CMV- and ‘non-specific’ CD8 + T-cells from HIV infected participants indicated in Table S2. B . Dimensionality reduction using tSNE on scRNA-Seq cells coloured by Louvain cluster (top), participant ID (middle), and tetramer specificity (bottom). C . Heatmap of z-scored gene expression of top differentially expressed genes (t-test) between Louvain clusters from scRNA-seq data with cells grouped by Louvain cluster, genes grouped by hierarchical clustering (full gene lists in supplemental Table S3). D . Gene set enrichment scores for each of the 4 Louvain clusters (0-3) shown for ‘ T RM ’ , ‘Exhaustion’ , ‘proliferation’ and ‘activation’ [ , ] published gene lists. E . Heatmaps of z-scored gene expression of top differentially expressed genes (t-test) between single cells with high and low normalized MFI values of CD69, CD103, PD-1 and CD127. Selected genes labelled in plot, full gene lists in Table S4-S5. F . Gene lists from (E) scored against the published gene lists as indicated in D.

Article Snippet: Exactly 4μm sections were cut, deparaffinized and stained with the following unlabelled primary antibodies: CD8 (clone: C8/144B, Dako), CD4 (clone: 4B12, Dako), p24 (clone: Kal-1, Dako), CXCR5 (clone: MU5UBEE, Thermofisher Scientific), Granzyme B (clone: 23 H8L20, Thermofisher Scientific), CD103 (clone: NBP1-88142, Novus Biologicals) and CD69 (clone: 15B5G2, Novus Biologicals).

Techniques: RNA Sequencing, Infection, Isolation, Gene Expression, Activation Assay