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Becton Dickinson bd facsaria ii
Adjuvanticity of FlgE to Soluble Antigen in Mice. ( A–C ) Naïve OVA-specific T cells were purified from the spleens and inguinal lymph nodes of CD45.1 × OTII F1 mice by sorting using various antibodies to surface markers, labeled with eFluor450 and transferred into female C57BL/6 mice at 1 × 10 6 cells/mouse via tail vein injection. Twenty-four hours later, the recipients were treated or immunized via subcutaneous injection at the base of the tail with one of eight compositions, namely 1 μg OVA, 50 μg CpG-1826, 50 μg FlgE, 50 μg FlgEM each alone, or OVA plus the three stimulants individually. Control groups received 100 μL PBS. Three days later, the mice were sacrificed, and the draining inguinal lymph nodes were isolated and photographed ( A ). Single-cell suspension were made for staining with CD45.1 plus CD4 and then run on a BD <t>FACSAria</t> II and analyzed for eFluor450 intensity to quantify the proliferation of OVA-specific T cells ( B,C ). ( D ) To measure the humoral response, WT C57BL/6 mice were immunized with above 8 compositions except for increasing OVA doses to 100 μg. Two weeks later, the mice were sacrificed for serum harvest, and anti-OVA titers were measured using an ELISA as described in Materials and Methods. Data are representatives of two independent experiments that showed similar results. n = 3 mice for panel A–C, and n = 4 mice for panel D. *P
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1) Product Images from "Flagellar Hooks and Hook Protein FlgE Participate in Host Microbe Interactions at Immunological Level"

Article Title: Flagellar Hooks and Hook Protein FlgE Participate in Host Microbe Interactions at Immunological Level

Journal: Scientific Reports

doi: 10.1038/s41598-017-01619-1

Adjuvanticity of FlgE to Soluble Antigen in Mice. ( A–C ) Naïve OVA-specific T cells were purified from the spleens and inguinal lymph nodes of CD45.1 × OTII F1 mice by sorting using various antibodies to surface markers, labeled with eFluor450 and transferred into female C57BL/6 mice at 1 × 10 6 cells/mouse via tail vein injection. Twenty-four hours later, the recipients were treated or immunized via subcutaneous injection at the base of the tail with one of eight compositions, namely 1 μg OVA, 50 μg CpG-1826, 50 μg FlgE, 50 μg FlgEM each alone, or OVA plus the three stimulants individually. Control groups received 100 μL PBS. Three days later, the mice were sacrificed, and the draining inguinal lymph nodes were isolated and photographed ( A ). Single-cell suspension were made for staining with CD45.1 plus CD4 and then run on a BD FACSAria II and analyzed for eFluor450 intensity to quantify the proliferation of OVA-specific T cells ( B,C ). ( D ) To measure the humoral response, WT C57BL/6 mice were immunized with above 8 compositions except for increasing OVA doses to 100 μg. Two weeks later, the mice were sacrificed for serum harvest, and anti-OVA titers were measured using an ELISA as described in Materials and Methods. Data are representatives of two independent experiments that showed similar results. n = 3 mice for panel A–C, and n = 4 mice for panel D. *P
Figure Legend Snippet: Adjuvanticity of FlgE to Soluble Antigen in Mice. ( A–C ) Naïve OVA-specific T cells were purified from the spleens and inguinal lymph nodes of CD45.1 × OTII F1 mice by sorting using various antibodies to surface markers, labeled with eFluor450 and transferred into female C57BL/6 mice at 1 × 10 6 cells/mouse via tail vein injection. Twenty-four hours later, the recipients were treated or immunized via subcutaneous injection at the base of the tail with one of eight compositions, namely 1 μg OVA, 50 μg CpG-1826, 50 μg FlgE, 50 μg FlgEM each alone, or OVA plus the three stimulants individually. Control groups received 100 μL PBS. Three days later, the mice were sacrificed, and the draining inguinal lymph nodes were isolated and photographed ( A ). Single-cell suspension were made for staining with CD45.1 plus CD4 and then run on a BD FACSAria II and analyzed for eFluor450 intensity to quantify the proliferation of OVA-specific T cells ( B,C ). ( D ) To measure the humoral response, WT C57BL/6 mice were immunized with above 8 compositions except for increasing OVA doses to 100 μg. Two weeks later, the mice were sacrificed for serum harvest, and anti-OVA titers were measured using an ELISA as described in Materials and Methods. Data are representatives of two independent experiments that showed similar results. n = 3 mice for panel A–C, and n = 4 mice for panel D. *P

Techniques Used: Mouse Assay, Purification, Labeling, Injection, Isolation, Staining, Enzyme-linked Immunosorbent Assay

2) Product Images from "Higher-Order Clustering of the Transmembrane Anchor of DR5 Drives Signaling"

Article Title: Higher-Order Clustering of the Transmembrane Anchor of DR5 Drives Signaling

Journal: Cell

doi: 10.1016/j.cell.2019.02.001

Higher-Order Clustering is Inhibited by the Unliganded Extracellular Domain ( A ) Schematic illustration of possible mechanism underlying proteolytic activation of DR5. ( B )Comparing TRAIL sensitivity of the DR5-TEV-expressing BJAB cells to that of the DR5-WT-expressing BJAB cells. Caspase-8 activity was measured using the CaspGLOW red Caspase-8 activity kit (Biovision) and flow cytometry (BD FACSAria II) and was calculated as percentage of DsRed2+/GFP+ cells divided by percentage of GFP+ cells. Results were from 3 independent experiments (n=3), and expressed as mean ± SEM. ( C )TEV-induced apoptosis. DR5-deficient BJAB cells were transiently transfected with WT DR5-EGFP with (DR5-TEV) or without (DR5-WT) TEV cleavage site between the ECD and TMD. After transfection, BJAB cells were treated with TEV for 5 h before analysis. ( D )Examples of CFP and YFP photobleaching for WT DR5 without (left) and with (right) TRAIL. ( E )Receptor self-association of the WT DR5 and mutants. The effects of dimer-breaking mutation (G217Y) and trimer-breaking mutation (A222Y) with or without TRAIL pre-treatment are quantitated in the form of FRET efficiency. CFP/YFP co-transfected cells were used as FRET negative control. N.S indicates not significant. Twelve regions of interest (n=12) (ROI, e.g., plasma membrane bound DR5) from different cells were examined by FRET study. .
Figure Legend Snippet: Higher-Order Clustering is Inhibited by the Unliganded Extracellular Domain ( A ) Schematic illustration of possible mechanism underlying proteolytic activation of DR5. ( B )Comparing TRAIL sensitivity of the DR5-TEV-expressing BJAB cells to that of the DR5-WT-expressing BJAB cells. Caspase-8 activity was measured using the CaspGLOW red Caspase-8 activity kit (Biovision) and flow cytometry (BD FACSAria II) and was calculated as percentage of DsRed2+/GFP+ cells divided by percentage of GFP+ cells. Results were from 3 independent experiments (n=3), and expressed as mean ± SEM. ( C )TEV-induced apoptosis. DR5-deficient BJAB cells were transiently transfected with WT DR5-EGFP with (DR5-TEV) or without (DR5-WT) TEV cleavage site between the ECD and TMD. After transfection, BJAB cells were treated with TEV for 5 h before analysis. ( D )Examples of CFP and YFP photobleaching for WT DR5 without (left) and with (right) TRAIL. ( E )Receptor self-association of the WT DR5 and mutants. The effects of dimer-breaking mutation (G217Y) and trimer-breaking mutation (A222Y) with or without TRAIL pre-treatment are quantitated in the form of FRET efficiency. CFP/YFP co-transfected cells were used as FRET negative control. N.S indicates not significant. Twelve regions of interest (n=12) (ROI, e.g., plasma membrane bound DR5) from different cells were examined by FRET study. .

Techniques Used: Activation Assay, Expressing, Activity Assay, Flow Cytometry, Transfection, Mutagenesis, Negative Control

3) Product Images from "Flagellar Hooks and Hook Protein FlgE Participate in Host Microbe Interactions at Immunological Level"

Article Title: Flagellar Hooks and Hook Protein FlgE Participate in Host Microbe Interactions at Immunological Level

Journal: Scientific Reports

doi: 10.1038/s41598-017-01619-1

Adjuvanticity of FlgE to Soluble Antigen in Mice. ( A–C ) Naïve OVA-specific T cells were purified from the spleens and inguinal lymph nodes of CD45.1 × OTII F1 mice by sorting using various antibodies to surface markers, labeled with eFluor450 and transferred into female C57BL/6 mice at 1 × 10 6 cells/mouse via tail vein injection. Twenty-four hours later, the recipients were treated or immunized via subcutaneous injection at the base of the tail with one of eight compositions, namely 1 μg OVA, 50 μg CpG-1826, 50 μg FlgE, 50 μg FlgEM each alone, or OVA plus the three stimulants individually. Control groups received 100 μL PBS. Three days later, the mice were sacrificed, and the draining inguinal lymph nodes were isolated and photographed ( A ). Single-cell suspension were made for staining with CD45.1 plus CD4 and then run on a BD FACSAria II and analyzed for eFluor450 intensity to quantify the proliferation of OVA-specific T cells ( B,C ). ( D ) To measure the humoral response, WT C57BL/6 mice were immunized with above 8 compositions except for increasing OVA doses to 100 μg. Two weeks later, the mice were sacrificed for serum harvest, and anti-OVA titers were measured using an ELISA as described in Materials and Methods. Data are representatives of two independent experiments that showed similar results. n = 3 mice for panel A–C, and n = 4 mice for panel D. *P
Figure Legend Snippet: Adjuvanticity of FlgE to Soluble Antigen in Mice. ( A–C ) Naïve OVA-specific T cells were purified from the spleens and inguinal lymph nodes of CD45.1 × OTII F1 mice by sorting using various antibodies to surface markers, labeled with eFluor450 and transferred into female C57BL/6 mice at 1 × 10 6 cells/mouse via tail vein injection. Twenty-four hours later, the recipients were treated or immunized via subcutaneous injection at the base of the tail with one of eight compositions, namely 1 μg OVA, 50 μg CpG-1826, 50 μg FlgE, 50 μg FlgEM each alone, or OVA plus the three stimulants individually. Control groups received 100 μL PBS. Three days later, the mice were sacrificed, and the draining inguinal lymph nodes were isolated and photographed ( A ). Single-cell suspension were made for staining with CD45.1 plus CD4 and then run on a BD FACSAria II and analyzed for eFluor450 intensity to quantify the proliferation of OVA-specific T cells ( B,C ). ( D ) To measure the humoral response, WT C57BL/6 mice were immunized with above 8 compositions except for increasing OVA doses to 100 μg. Two weeks later, the mice were sacrificed for serum harvest, and anti-OVA titers were measured using an ELISA as described in Materials and Methods. Data are representatives of two independent experiments that showed similar results. n = 3 mice for panel A–C, and n = 4 mice for panel D. *P

Techniques Used: Mouse Assay, Purification, Labeling, Injection, Isolation, Staining, Enzyme-linked Immunosorbent Assay

4) Product Images from "Enhanced cancer stem cell properties of a mitotically quiescent subpopulation of p75NTR-positive cells in esophageal squamous cell carcinoma"

Article Title: Enhanced cancer stem cell properties of a mitotically quiescent subpopulation of p75NTR-positive cells in esophageal squamous cell carcinoma

Journal: International Journal of Oncology

doi: 10.3892/ijo.2017.4001

Flow cytometric cell sorting based on p75NTR expression and cell cycle status. (A) The left panels show representative results of double immunocytochemical staining of Ki-67 (brown) and p75NTR (red) in KYSE-30 (upper panel) and KYSE-140 cells (lower panel). Nuclei are stained with hematoxylin. The white, red, blue and green arrowheads indicate p75NTR-positive/Ki-67-negative, p75NTR-positive/Ki-67-positive, p75NTR-negative/Ki-67-negative and p75NTR-negative/Ki-67-positive cells, respectively. The middle panels show representative results of flow cytometric analysis of p75NTR expression in KYSE-30 (upper panel) and KYSE-140 cells (lower panel). Cut-off levels were set based on control IgG, and percentages of positive cells are shown in the panels. The right panels show cell cycle distribution in p75NTR-positive and p75NTR-negative KYSE-30 (upper panel) and KYSE-140 cells (lower panel). Live cells were analyzed using a DNA-staining dye (DCV) and FACSAria II. The proportion of cells in each phase of the cell cycle is shown. (B) Diagram showing the steps of flow cytometric cell fractionation based on p75NTR expression and cell cycle status. KYSE-30 and KYSE-140 cells were fractionated into p75NTR-positive and p75NTR-negative cells. Next, each cell subset was fractionated into cells in the G0-G1 and S-G2-M phases of the cell cycle by using a fluorescent DNA-staining dye (DCV). (C) The left panels show p75NTR expression in the four cell subsets sorted from KYSE-30 (upper panel) and KYSE-140 cells (lower panel), as determined by performing real-time PCR. The right panels show cell cycle status of each of the four cell subsets sorted from KYSE-30 (upper panel) and KYSE-140 cells (lower panel), as determined using another fluorescent DNA-staining dye (BD CycleTest™ Plus DNA reagent kit). The error bars represent standard error of mean; *** P
Figure Legend Snippet: Flow cytometric cell sorting based on p75NTR expression and cell cycle status. (A) The left panels show representative results of double immunocytochemical staining of Ki-67 (brown) and p75NTR (red) in KYSE-30 (upper panel) and KYSE-140 cells (lower panel). Nuclei are stained with hematoxylin. The white, red, blue and green arrowheads indicate p75NTR-positive/Ki-67-negative, p75NTR-positive/Ki-67-positive, p75NTR-negative/Ki-67-negative and p75NTR-negative/Ki-67-positive cells, respectively. The middle panels show representative results of flow cytometric analysis of p75NTR expression in KYSE-30 (upper panel) and KYSE-140 cells (lower panel). Cut-off levels were set based on control IgG, and percentages of positive cells are shown in the panels. The right panels show cell cycle distribution in p75NTR-positive and p75NTR-negative KYSE-30 (upper panel) and KYSE-140 cells (lower panel). Live cells were analyzed using a DNA-staining dye (DCV) and FACSAria II. The proportion of cells in each phase of the cell cycle is shown. (B) Diagram showing the steps of flow cytometric cell fractionation based on p75NTR expression and cell cycle status. KYSE-30 and KYSE-140 cells were fractionated into p75NTR-positive and p75NTR-negative cells. Next, each cell subset was fractionated into cells in the G0-G1 and S-G2-M phases of the cell cycle by using a fluorescent DNA-staining dye (DCV). (C) The left panels show p75NTR expression in the four cell subsets sorted from KYSE-30 (upper panel) and KYSE-140 cells (lower panel), as determined by performing real-time PCR. The right panels show cell cycle status of each of the four cell subsets sorted from KYSE-30 (upper panel) and KYSE-140 cells (lower panel), as determined using another fluorescent DNA-staining dye (BD CycleTest™ Plus DNA reagent kit). The error bars represent standard error of mean; *** P

Techniques Used: Flow Cytometry, FACS, Expressing, Staining, Cell Fractionation, Real-time Polymerase Chain Reaction

5) Product Images from "Ex Vivo Expansion of Murine MSC Impairs Transcription Factor-Induced Differentiation into Pancreatic β-Cells"

Article Title: Ex Vivo Expansion of Murine MSC Impairs Transcription Factor-Induced Differentiation into Pancreatic β-Cells

Journal: Stem Cells International

doi: 10.1155/2019/1395301

In vitro characteristics of NOD-derived MSCs with cell culture expansion. (a) FACS analysis and enrichment of NOD-derived MSCs. Following culture for two passages, NOD bone marrow stromal cells were stained with nil antibody (unstained), CD45 mAb conjugated to fluorochrome APC (CD45-APC), Ly6 MAb conjugated to fluorochrome PE (Ly6-PE), and both mAbs (CD45-APC/Ly6-PE). Fluorescence dot plots of CD45-APC ( y -axis) and Ly6-PE ( x -axis) were used to identify the MSC (CD45 − /Ly6 + ; orange) and double-positive (CD45 + /Ly6 + ; red) cell subpopulations ready for cell sorting using the BD FACSAria™ II instrument. Representative of three individual FACS experiments. (b) Plastic adherence, fibroblast-like morphology, and self-renewal without differentiation into other cell types. MSCs maintained fibroblast-like morphology as assessed using light microscopy (Leica DM microscope; 10x magnification; scale bar = 100 μ M). (c) Improved cell proliferation with culture expansion. Data are presented as mean viable cells ± SDs ( n = 3). A two-way ANOVA with Tukey's posttests was performed, ∗ p
Figure Legend Snippet: In vitro characteristics of NOD-derived MSCs with cell culture expansion. (a) FACS analysis and enrichment of NOD-derived MSCs. Following culture for two passages, NOD bone marrow stromal cells were stained with nil antibody (unstained), CD45 mAb conjugated to fluorochrome APC (CD45-APC), Ly6 MAb conjugated to fluorochrome PE (Ly6-PE), and both mAbs (CD45-APC/Ly6-PE). Fluorescence dot plots of CD45-APC ( y -axis) and Ly6-PE ( x -axis) were used to identify the MSC (CD45 − /Ly6 + ; orange) and double-positive (CD45 + /Ly6 + ; red) cell subpopulations ready for cell sorting using the BD FACSAria™ II instrument. Representative of three individual FACS experiments. (b) Plastic adherence, fibroblast-like morphology, and self-renewal without differentiation into other cell types. MSCs maintained fibroblast-like morphology as assessed using light microscopy (Leica DM microscope; 10x magnification; scale bar = 100 μ M). (c) Improved cell proliferation with culture expansion. Data are presented as mean viable cells ± SDs ( n = 3). A two-way ANOVA with Tukey's posttests was performed, ∗ p

Techniques Used: In Vitro, Derivative Assay, Cell Culture, FACS, Staining, Fluorescence, Light Microscopy, Microscopy

6) Product Images from "The golgin family exhibits a propensity to form condensates in living cells"

Article Title: The golgin family exhibits a propensity to form condensates in living cells

Journal: Febs Letters

doi: 10.1002/1873-3468.13884

FIB‐SEM of GM130 and golgin160 condensates. HeLa cells were transfected with (A) GFP‐GM130 for 20 h or (B) golgin160‐GFP for 10 h, enriched for GFP + cells using a BD FACSAria II cell sorter, and visualized using FIB‐SEM. GM130 condensate in (A) is seen as a proteinaceous sphere (black) riddled with aqueous channels (white). Golgin160 condensates in (B) are indicated by arrowheads. The frame of the FIB‐SEM tomogram is indicated in the top left corner of each panel. Scale bars are 500 nm for (A) and 1 µm for (B).
Figure Legend Snippet: FIB‐SEM of GM130 and golgin160 condensates. HeLa cells were transfected with (A) GFP‐GM130 for 20 h or (B) golgin160‐GFP for 10 h, enriched for GFP + cells using a BD FACSAria II cell sorter, and visualized using FIB‐SEM. GM130 condensate in (A) is seen as a proteinaceous sphere (black) riddled with aqueous channels (white). Golgin160 condensates in (B) are indicated by arrowheads. The frame of the FIB‐SEM tomogram is indicated in the top left corner of each panel. Scale bars are 500 nm for (A) and 1 µm for (B).

Techniques Used: Transfection

7) Product Images from "Tasquinimod modulates tumor-infiltrating myeloid cells and improves the antitumor immune response to PD-L1 blockade in bladder cancer"

Article Title: Tasquinimod modulates tumor-infiltrating myeloid cells and improves the antitumor immune response to PD-L1 blockade in bladder cancer

Journal: Oncoimmunology

doi: 10.1080/2162402X.2016.1145333

Combining a modulator of infiltrating-myeloid cells and an inhibitor of PD-1/PD-L1 axis increases T cell proliferation and T cell producing IFNγ. Myeloid cells CD11b+ were isolated from tumors using BD FACSAria II (BD Biosciences). T cells were isolated from spleen of naive mice using mouse pan T cell isolation Kit (Miltenyei). CFSE-labeled T cells were stimulated with CD3/CD28 beads ratio 1:1 (Life Technologies). Stimulated T cells were cultured with CD11b+ (at a ratio CD11b:T cells of 1:1) and incubated for 72 h at 37°C. (A) Representative histograms obtained by FACS analysis showing the fluorescence intensity of CFSE-T cells gated on CD8 + . (B) The percentage of proliferating CD8 + cells from the different treated groups is shown. (C) IFNγ secretion in the supernatant of the co-culture is measured 72 h following incubation at 37°C using Luminex Technology. Experiments were repeated twice (Kruskal–Wallis test, * p
Figure Legend Snippet: Combining a modulator of infiltrating-myeloid cells and an inhibitor of PD-1/PD-L1 axis increases T cell proliferation and T cell producing IFNγ. Myeloid cells CD11b+ were isolated from tumors using BD FACSAria II (BD Biosciences). T cells were isolated from spleen of naive mice using mouse pan T cell isolation Kit (Miltenyei). CFSE-labeled T cells were stimulated with CD3/CD28 beads ratio 1:1 (Life Technologies). Stimulated T cells were cultured with CD11b+ (at a ratio CD11b:T cells of 1:1) and incubated for 72 h at 37°C. (A) Representative histograms obtained by FACS analysis showing the fluorescence intensity of CFSE-T cells gated on CD8 + . (B) The percentage of proliferating CD8 + cells from the different treated groups is shown. (C) IFNγ secretion in the supernatant of the co-culture is measured 72 h following incubation at 37°C using Luminex Technology. Experiments were repeated twice (Kruskal–Wallis test, * p

Techniques Used: Isolation, Mouse Assay, Cell Isolation, Labeling, Cell Culture, Incubation, FACS, Fluorescence, Co-Culture Assay, Luminex

8) Product Images from "Insertional Mutagenesis by CRISPR/Cas9 Ribonucleoprotein Gene Editing in Cells Targeted for Point Mutation Repair Directed by Short Single-Stranded DNA Oligonucleotides"

Article Title: Insertional Mutagenesis by CRISPR/Cas9 Ribonucleoprotein Gene Editing in Cells Targeted for Point Mutation Repair Directed by Short Single-Stranded DNA Oligonucleotides

Journal: PLoS ONE

doi: 10.1371/journal.pone.0169350

(A) Gene editing is dose dependent when directed by the RNP and the ssODN. Synchronized and released HCT 116–19 cells were electroporated with 24–120 pmol CRISPR/Cas9 RNP and 0.6–3.0 μM of 72mer. After a 72-hour recovery period, gene editing activity was measured using a FACSAria II flow cytometer. Gene editing is displayed as correction efficiency (%), determined by the number of viable eGFP positive cells divided by the total number of viable cells in the population. Each treatment was performed in triplicate and standard error is illustrated with accompanying bars. Inset: Single agent gene editing . Gene editing activity directed by the single-stranded oligonucleotide (72NT) in the absence of the RNP complex under identical conditions is presented as a function of increasing concentration. (B) Gene editing activity is dependent on all components being present in the reaction mixture. Synchronized and released HCT 116–19 cells were electroporated with 100pmol of the crRNA, Cas9 Protein, tracrRNA and 2.0 μM of the 72NT, as a complete reaction. Identical mixtures, lacking the indicated reaction component, were carried out in parallel. In one specific reaction mixture, the RNP specific for the beta globin gene replaced the RNP specific for the eGFP gene (far right bar). After a 72 hour recovery period, gene editing activity was measured using a FACSAria II flow cytometer. Gene editing is displayed as correction efficiency (%), determined by the number of viable eGFP positive cells divided by the total number of viable cells in the population. Each treatment was performed in triplicate and standard error is illustrated with accompanying bars.
Figure Legend Snippet: (A) Gene editing is dose dependent when directed by the RNP and the ssODN. Synchronized and released HCT 116–19 cells were electroporated with 24–120 pmol CRISPR/Cas9 RNP and 0.6–3.0 μM of 72mer. After a 72-hour recovery period, gene editing activity was measured using a FACSAria II flow cytometer. Gene editing is displayed as correction efficiency (%), determined by the number of viable eGFP positive cells divided by the total number of viable cells in the population. Each treatment was performed in triplicate and standard error is illustrated with accompanying bars. Inset: Single agent gene editing . Gene editing activity directed by the single-stranded oligonucleotide (72NT) in the absence of the RNP complex under identical conditions is presented as a function of increasing concentration. (B) Gene editing activity is dependent on all components being present in the reaction mixture. Synchronized and released HCT 116–19 cells were electroporated with 100pmol of the crRNA, Cas9 Protein, tracrRNA and 2.0 μM of the 72NT, as a complete reaction. Identical mixtures, lacking the indicated reaction component, were carried out in parallel. In one specific reaction mixture, the RNP specific for the beta globin gene replaced the RNP specific for the eGFP gene (far right bar). After a 72 hour recovery period, gene editing activity was measured using a FACSAria II flow cytometer. Gene editing is displayed as correction efficiency (%), determined by the number of viable eGFP positive cells divided by the total number of viable cells in the population. Each treatment was performed in triplicate and standard error is illustrated with accompanying bars.

Techniques Used: CRISPR, Activity Assay, Flow Cytometry, Cytometry, Concentration Assay

(A) FACSAria II plots of gene editing activity in HCT 116–19 cells . HCT 116–19 cells synchronized for 24 hours at the G1/S border and released were electroporated with 100 pmol of RNP complex and 2.0 μM of the 72NT ssODN. After 72 hours, the cells were analyzed using FACS and single cells were sorted individually into 96-well plates. Two distinct populations were collected. The population of live, green cells (labeled as P2 on the FACS plot) as well as the population of live, non-green cells (labeled as P3) were segregated into separate clonal expansion plates. (B) Experimental strategy isolation of single cell clones. Cells exhibiting eGFP expression were scored positive and sorted using a FACSAria II flow cytometer as single cells into individual wells for clonal expansion. Cells lacking eGFP expression isolated and sorted in a similar fashion and expanded under the same conditions. DNA was then isolated and the eGFP gene was amplified and subjected to Sanger sequencing to analyze gene editing activity surrounding the target site.
Figure Legend Snippet: (A) FACSAria II plots of gene editing activity in HCT 116–19 cells . HCT 116–19 cells synchronized for 24 hours at the G1/S border and released were electroporated with 100 pmol of RNP complex and 2.0 μM of the 72NT ssODN. After 72 hours, the cells were analyzed using FACS and single cells were sorted individually into 96-well plates. Two distinct populations were collected. The population of live, green cells (labeled as P2 on the FACS plot) as well as the population of live, non-green cells (labeled as P3) were segregated into separate clonal expansion plates. (B) Experimental strategy isolation of single cell clones. Cells exhibiting eGFP expression were scored positive and sorted using a FACSAria II flow cytometer as single cells into individual wells for clonal expansion. Cells lacking eGFP expression isolated and sorted in a similar fashion and expanded under the same conditions. DNA was then isolated and the eGFP gene was amplified and subjected to Sanger sequencing to analyze gene editing activity surrounding the target site.

Techniques Used: Activity Assay, FACS, Labeling, Isolation, Clone Assay, Expressing, Flow Cytometry, Cytometry, Amplification, Sequencing

9) Product Images from "The Combinations Chitosan-Pam3CSK4 and Chitosan-Monophosphoryl Lipid A: Promising Immune-Enhancing Adjuvants for Anticaries Vaccine PAc"

Article Title: The Combinations Chitosan-Pam3CSK4 and Chitosan-Monophosphoryl Lipid A: Promising Immune-Enhancing Adjuvants for Anticaries Vaccine PAc

Journal: Infection and Immunity

doi: 10.1128/IAI.00651-19

Adjuvanticity of MDP, chitosan, Pam 3 CSK 4 , MPL, and iE-DAP to OVA in OVA-specific TCR transgenic mice. Naive OVA-specific T cells were purified from the spleens and inguinal lymph nodes of CD45.1×OTII F 1 mice and transferred into female C57BL/6J mice. One day later, the recipients were immunized via buccal mucosal injection with one of seven compositions in 40 μl, namely, 1 μg of OVA, OVA plus 40 μg of MDP, OVA plus 32 μg of chitosan, OVA plus 25 μg of Pam 3 CSK 4 , OVA plus 15 μg of MPL, OVA plus 50 μg of iE-DAP, or PBS control. Three days later, the mice were sacrificed. The draining submandibular lymph nodes were isolated, and the cells were counted (A). Single-cell suspensions were made for staining with CD45.1 plus CD4 and then run on a BD FACSAria II and analyzed for the number of OVA-specific T cells (B). *, Significantly different from the PBS group (*, P
Figure Legend Snippet: Adjuvanticity of MDP, chitosan, Pam 3 CSK 4 , MPL, and iE-DAP to OVA in OVA-specific TCR transgenic mice. Naive OVA-specific T cells were purified from the spleens and inguinal lymph nodes of CD45.1×OTII F 1 mice and transferred into female C57BL/6J mice. One day later, the recipients were immunized via buccal mucosal injection with one of seven compositions in 40 μl, namely, 1 μg of OVA, OVA plus 40 μg of MDP, OVA plus 32 μg of chitosan, OVA plus 25 μg of Pam 3 CSK 4 , OVA plus 15 μg of MPL, OVA plus 50 μg of iE-DAP, or PBS control. Three days later, the mice were sacrificed. The draining submandibular lymph nodes were isolated, and the cells were counted (A). Single-cell suspensions were made for staining with CD45.1 plus CD4 and then run on a BD FACSAria II and analyzed for the number of OVA-specific T cells (B). *, Significantly different from the PBS group (*, P

Techniques Used: Transgenic Assay, Mouse Assay, Purification, Injection, Isolation, Staining

Adjuvanticity of MDP, chitosan, Pam 3 CSK 4 , MPL and iE-DAP to OVA in C57BL/6J mice.The C57BL/6J mice were immunized on day 0 via buccal mucosal injection with 100 μg of OVA, OVA plus 40 μg of MDP, OVA plus 32 μg of chitosan, OVA plus 25 μg of Pam 3 CSK 4 , OVA plus 15 μg of MPL, OVA plus 50 μg of iE-DAP, or PBS and boosted on day 7. On day 14, the sera were collected for measuring OVA-specific IgG1 and IgG2b responses (see panel E). On day 15, the mice were administered with 100 μg of OVA via buccal mucosal injection. On day 17, the mice were sacrificed; the draining submandibular lymph nodes were isolated (A), and the cells were counted (B). (C and D) Single-cell suspensions were made for staining with CD4 plus CD8 and then run on a BD FACSAria II and analyzed for the numbers of CD4 + and CD8 + T cells. C57BL/6J mice were immunized with 100 μg of OVA, OVA plus 32 μg of chitosan, OVA plus chitosan plus 15 μg of MPL, or OVA plus chitosan plus 25 μg of Pam 3 CSK 4 on day 0 and boosted on day 7, all via buccal mucosa injection. On day 14, the mice were sacrificed, and sera were harvested to measure the OVA-specific IgG1 and IgG2b responses (F). *, Significantly different from the PBS group (*, P
Figure Legend Snippet: Adjuvanticity of MDP, chitosan, Pam 3 CSK 4 , MPL and iE-DAP to OVA in C57BL/6J mice.The C57BL/6J mice were immunized on day 0 via buccal mucosal injection with 100 μg of OVA, OVA plus 40 μg of MDP, OVA plus 32 μg of chitosan, OVA plus 25 μg of Pam 3 CSK 4 , OVA plus 15 μg of MPL, OVA plus 50 μg of iE-DAP, or PBS and boosted on day 7. On day 14, the sera were collected for measuring OVA-specific IgG1 and IgG2b responses (see panel E). On day 15, the mice were administered with 100 μg of OVA via buccal mucosal injection. On day 17, the mice were sacrificed; the draining submandibular lymph nodes were isolated (A), and the cells were counted (B). (C and D) Single-cell suspensions were made for staining with CD4 plus CD8 and then run on a BD FACSAria II and analyzed for the numbers of CD4 + and CD8 + T cells. C57BL/6J mice were immunized with 100 μg of OVA, OVA plus 32 μg of chitosan, OVA plus chitosan plus 15 μg of MPL, or OVA plus chitosan plus 25 μg of Pam 3 CSK 4 on day 0 and boosted on day 7, all via buccal mucosa injection. On day 14, the mice were sacrificed, and sera were harvested to measure the OVA-specific IgG1 and IgG2b responses (F). *, Significantly different from the PBS group (*, P

Techniques Used: Mouse Assay, Injection, Isolation, Staining

10) Product Images from "Membrane-proximal external region is a superior target for mediating effector activity of HIV-1 specific chimeric antigen receptor modified T cells"

Article Title: Membrane-proximal external region is a superior target for mediating effector activity of HIV-1 specific chimeric antigen receptor modified T cells

Journal: bioRxiv

doi: 10.1101/2020.03.11.987610

Generation of Jurkat cells inducibly expressing envelope protein from HIV-1 HXBC2 (HXBC2). (A) Schematic of lentiviral vectors for expressing TagBFP and mCherry. These vectors have an FG12-derived backbone possessing a self-inactivating LTR, central polypurine tract (cPPT), ubiquitin C promoter (UbiC), and a mutant Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE). (B) Jurkat cells with or without inducibly expressing envelope protein from HIV-1 HXBC2 first had human-β2 microglobulin (β2MG) expression knocked out by CRISPR-Cas9 gene editing using a non-integrating lentiviral vector encoding Cas9 together with sgRNA specific for β2MG as shown in S1 Fig . Populations missing human-leukocyte antigen (HLA)-1 A, B, and C expression were negatively enriched by magnetic bead separation, followed by transduction with a lentiviral vector encoding either TagBFP for ΔKS or mCherry for HXBC2. Cells were then labeled by PE/Cy7-conjugated anti-human HLA-I A,B,C and BV711-conjugated human CD4 antibodies then further selected for TagBFP + /HLA-I A,B,C - /CD4 dim (ΔKS) or mCherry + /HLA-I A,B,C - /CD4 dim (HXBC2) populations by FACSAria II flow sorter. (C) Equal numbers of ΔKS, HXBC2, and unmodified Jurkat cells were mixed and analyzed on LSRFortessa for TagBFP and mCherry expression. (D) HXBC2 cells were cultured for 4 days in the presence or absence of 1 µg/ml doxycycline (DOX) to induce HIV-1 HXBC2 envelope expression. Five million cells were lysed in 1% CHAPS and HIV-1 envelope expression was analyzed by western blotting using a mixture of anti-HIV-1 gp41 (2F5) and anti-HIV-1 gp120 (2G12) antibodies. Numbers to the right of the picture indicate molecular mass in kilodaltons. +: with DOX, -: without DOX. (E) A fusion protein of soluble human CD4 and Fc portion of human IgG1 (sCD4 Fc) was used for detection of HIV-1 envelope expression on cell surface. One million HXBC2 cells cultured in the absence (red) or presence (blue) of DOX for 6, 9, and 12 days were incubated with 1 µg sCD4 Fc on ice, followed by APC-conjugated anti-human Ig Fc portion. Human primary T cells (two donors) were infected with HIV-1 AD8 and HIV-1 BaL and used as a positive control for levels of cell surface expression ofHIV-1 envelope proteins detected by sCD4 Fc. Gray: uninfected human primary T cells, Red: HIV-1-infected cells.
Figure Legend Snippet: Generation of Jurkat cells inducibly expressing envelope protein from HIV-1 HXBC2 (HXBC2). (A) Schematic of lentiviral vectors for expressing TagBFP and mCherry. These vectors have an FG12-derived backbone possessing a self-inactivating LTR, central polypurine tract (cPPT), ubiquitin C promoter (UbiC), and a mutant Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE). (B) Jurkat cells with or without inducibly expressing envelope protein from HIV-1 HXBC2 first had human-β2 microglobulin (β2MG) expression knocked out by CRISPR-Cas9 gene editing using a non-integrating lentiviral vector encoding Cas9 together with sgRNA specific for β2MG as shown in S1 Fig . Populations missing human-leukocyte antigen (HLA)-1 A, B, and C expression were negatively enriched by magnetic bead separation, followed by transduction with a lentiviral vector encoding either TagBFP for ΔKS or mCherry for HXBC2. Cells were then labeled by PE/Cy7-conjugated anti-human HLA-I A,B,C and BV711-conjugated human CD4 antibodies then further selected for TagBFP + /HLA-I A,B,C - /CD4 dim (ΔKS) or mCherry + /HLA-I A,B,C - /CD4 dim (HXBC2) populations by FACSAria II flow sorter. (C) Equal numbers of ΔKS, HXBC2, and unmodified Jurkat cells were mixed and analyzed on LSRFortessa for TagBFP and mCherry expression. (D) HXBC2 cells were cultured for 4 days in the presence or absence of 1 µg/ml doxycycline (DOX) to induce HIV-1 HXBC2 envelope expression. Five million cells were lysed in 1% CHAPS and HIV-1 envelope expression was analyzed by western blotting using a mixture of anti-HIV-1 gp41 (2F5) and anti-HIV-1 gp120 (2G12) antibodies. Numbers to the right of the picture indicate molecular mass in kilodaltons. +: with DOX, -: without DOX. (E) A fusion protein of soluble human CD4 and Fc portion of human IgG1 (sCD4 Fc) was used for detection of HIV-1 envelope expression on cell surface. One million HXBC2 cells cultured in the absence (red) or presence (blue) of DOX for 6, 9, and 12 days were incubated with 1 µg sCD4 Fc on ice, followed by APC-conjugated anti-human Ig Fc portion. Human primary T cells (two donors) were infected with HIV-1 AD8 and HIV-1 BaL and used as a positive control for levels of cell surface expression ofHIV-1 envelope proteins detected by sCD4 Fc. Gray: uninfected human primary T cells, Red: HIV-1-infected cells.

Techniques Used: Expressing, Derivative Assay, Mutagenesis, CRISPR, Plasmid Preparation, Transduction, Labeling, Cell Culture, Western Blot, Incubation, Infection, Positive Control

11) Product Images from "Unveiling a CD70-positive subset of cancer-associated fibroblasts marked by pro-migratory activity and thriving regulatory T cell accumulation"

Article Title: Unveiling a CD70-positive subset of cancer-associated fibroblasts marked by pro-migratory activity and thriving regulatory T cell accumulation

Journal: Oncoimmunology

doi: 10.1080/2162402X.2018.1440167

Role of CD70-positive CAFs on regulatory T cell accumulation. CT5.3 hTERT CAFs were sorted on the membrane expression of CD70 (see also Fig. S1). CD3 + CD4 + T-cells were cultured alone (Ctrl) or co-cultured with CD70 pos /CD70 neg CAFs for 7 days. Afterwards, lymphocytes were analysed with a multicolour panel on a BD FACSAria II system (see also Fig. S2 for gating strategy). A: Representative dot plots showing the percentage of naturally occurring Tregs (nTregs) (CD3 + CD4 + CD25 + CD127 low ) after 7 days of co-culturing with CD70 low (left) or CD70 high (right) CAFs; B: Percentage nTregs (left) within CD4 + T-cell subset and induced Tregs (iTreg) (right) within CD4 + CD25 − T-cell subset; C: Percentage CD25 + (left) and CD25 − (right) cells within CD27 + T cells. D: IL-2 (left) and TGF-ß (right) protein levels in supernatants, 5 days after (co-)culture. Experiments were run in parallel with CD4 + T-cells from four different donors. * P
Figure Legend Snippet: Role of CD70-positive CAFs on regulatory T cell accumulation. CT5.3 hTERT CAFs were sorted on the membrane expression of CD70 (see also Fig. S1). CD3 + CD4 + T-cells were cultured alone (Ctrl) or co-cultured with CD70 pos /CD70 neg CAFs for 7 days. Afterwards, lymphocytes were analysed with a multicolour panel on a BD FACSAria II system (see also Fig. S2 for gating strategy). A: Representative dot plots showing the percentage of naturally occurring Tregs (nTregs) (CD3 + CD4 + CD25 + CD127 low ) after 7 days of co-culturing with CD70 low (left) or CD70 high (right) CAFs; B: Percentage nTregs (left) within CD4 + T-cell subset and induced Tregs (iTreg) (right) within CD4 + CD25 − T-cell subset; C: Percentage CD25 + (left) and CD25 − (right) cells within CD27 + T cells. D: IL-2 (left) and TGF-ß (right) protein levels in supernatants, 5 days after (co-)culture. Experiments were run in parallel with CD4 + T-cells from four different donors. * P

Techniques Used: Expressing, Cell Culture, Co-Culture Assay

12) Product Images from "Tasquinimod modulates tumor-infiltrating myeloid cells and improves the antitumor immune response to PD-L1 blockade in bladder cancer"

Article Title: Tasquinimod modulates tumor-infiltrating myeloid cells and improves the antitumor immune response to PD-L1 blockade in bladder cancer

Journal: Oncoimmunology

doi: 10.1080/2162402X.2016.1145333

Combining a modulator of infiltrating-myeloid cells and an inhibitor of PD-1/PD-L1 axis increases T cell proliferation and T cell producing IFNγ. Myeloid cells CD11b+ were isolated from tumors using BD FACSAria II (BD Biosciences). T cells were isolated from spleen of naive mice using mouse pan T cell isolation Kit (Miltenyei). CFSE-labeled T cells were stimulated with CD3/CD28 beads ratio 1:1 (Life Technologies). Stimulated T cells were cultured with CD11b+ (at a ratio CD11b:T cells of 1:1) and incubated for 72 h at 37°C. (A) Representative histograms obtained by FACS analysis showing the fluorescence intensity of CFSE-T cells gated on CD8 + . (B) The percentage of proliferating CD8 + cells from the different treated groups is shown. (C) IFNγ secretion in the supernatant of the co-culture is measured 72 h following incubation at 37°C using Luminex Technology. Experiments were repeated twice (Kruskal–Wallis test, * p
Figure Legend Snippet: Combining a modulator of infiltrating-myeloid cells and an inhibitor of PD-1/PD-L1 axis increases T cell proliferation and T cell producing IFNγ. Myeloid cells CD11b+ were isolated from tumors using BD FACSAria II (BD Biosciences). T cells were isolated from spleen of naive mice using mouse pan T cell isolation Kit (Miltenyei). CFSE-labeled T cells were stimulated with CD3/CD28 beads ratio 1:1 (Life Technologies). Stimulated T cells were cultured with CD11b+ (at a ratio CD11b:T cells of 1:1) and incubated for 72 h at 37°C. (A) Representative histograms obtained by FACS analysis showing the fluorescence intensity of CFSE-T cells gated on CD8 + . (B) The percentage of proliferating CD8 + cells from the different treated groups is shown. (C) IFNγ secretion in the supernatant of the co-culture is measured 72 h following incubation at 37°C using Luminex Technology. Experiments were repeated twice (Kruskal–Wallis test, * p

Techniques Used: Isolation, Mouse Assay, Cell Isolation, Labeling, Cell Culture, Incubation, FACS, Fluorescence, Co-Culture Assay, Luminex

Related Articles

Expressing:

Article Title: MiR-23~27~24–mediated control of humoral immunity reveals a TOX-driven regulatory circuit in follicular helper T cell differentiation
Article Snippet: GFP+ cells were FACS-sorted 3 days after electroporation and injected immediately into the congenically marked recipient mice for the SMARTA cell transfer study. .. Gene expression profiling and ChIP-seq data analysis CD4+ CD25− CD44− naïve T (Tn ) cells, CD4+ CD25− CD44+ CXCR5− PSGL1hi TH 1 cells, CD4+ CD25− CD44+ CXCR5+ PSGL1int TFH cells, and CD4+ CD25− CD44+ CXCR5+ PSGL1lo GC-TFH cells in the spleen from LCMV-infected T-DKO mice, and WT controls were sorted on a FACSAria II cell sorter (BD Biosciences) followed by total RNA isolation using a miRNeasy Kit (QIAGEN). .. Poly-A RNA-seq was performed using three biological replicates for each cell population, similar to what was described previously ( ).

ChIP-sequencing:

Article Title: MiR-23~27~24–mediated control of humoral immunity reveals a TOX-driven regulatory circuit in follicular helper T cell differentiation
Article Snippet: GFP+ cells were FACS-sorted 3 days after electroporation and injected immediately into the congenically marked recipient mice for the SMARTA cell transfer study. .. Gene expression profiling and ChIP-seq data analysis CD4+ CD25− CD44− naïve T (Tn ) cells, CD4+ CD25− CD44+ CXCR5− PSGL1hi TH 1 cells, CD4+ CD25− CD44+ CXCR5+ PSGL1int TFH cells, and CD4+ CD25− CD44+ CXCR5+ PSGL1lo GC-TFH cells in the spleen from LCMV-infected T-DKO mice, and WT controls were sorted on a FACSAria II cell sorter (BD Biosciences) followed by total RNA isolation using a miRNeasy Kit (QIAGEN). .. Poly-A RNA-seq was performed using three biological replicates for each cell population, similar to what was described previously ( ).

Mouse Assay:

Article Title: MiR-23~27~24–mediated control of humoral immunity reveals a TOX-driven regulatory circuit in follicular helper T cell differentiation
Article Snippet: GFP+ cells were FACS-sorted 3 days after electroporation and injected immediately into the congenically marked recipient mice for the SMARTA cell transfer study. .. Gene expression profiling and ChIP-seq data analysis CD4+ CD25− CD44− naïve T (Tn ) cells, CD4+ CD25− CD44+ CXCR5− PSGL1hi TH 1 cells, CD4+ CD25− CD44+ CXCR5+ PSGL1int TFH cells, and CD4+ CD25− CD44+ CXCR5+ PSGL1lo GC-TFH cells in the spleen from LCMV-infected T-DKO mice, and WT controls were sorted on a FACSAria II cell sorter (BD Biosciences) followed by total RNA isolation using a miRNeasy Kit (QIAGEN). .. Poly-A RNA-seq was performed using three biological replicates for each cell population, similar to what was described previously ( ).

Isolation:

Article Title: MiR-23~27~24–mediated control of humoral immunity reveals a TOX-driven regulatory circuit in follicular helper T cell differentiation
Article Snippet: GFP+ cells were FACS-sorted 3 days after electroporation and injected immediately into the congenically marked recipient mice for the SMARTA cell transfer study. .. Gene expression profiling and ChIP-seq data analysis CD4+ CD25− CD44− naïve T (Tn ) cells, CD4+ CD25− CD44+ CXCR5− PSGL1hi TH 1 cells, CD4+ CD25− CD44+ CXCR5+ PSGL1int TFH cells, and CD4+ CD25− CD44+ CXCR5+ PSGL1lo GC-TFH cells in the spleen from LCMV-infected T-DKO mice, and WT controls were sorted on a FACSAria II cell sorter (BD Biosciences) followed by total RNA isolation using a miRNeasy Kit (QIAGEN). .. Poly-A RNA-seq was performed using three biological replicates for each cell population, similar to what was described previously ( ).

FACS:

Article Title: Liver X receptors are required for thymic resilience and T cell output
Article Snippet: Data were acquired on an LSRII (BD Biosciences) and analyzed with FlowJo software. .. Cell sorting TECs and thymocytes underwent FACS using a FACSAria II cell sorter (BD Biosciences) with 7-AAD used for cell viability. .. For bulk TEC sorting, samples were stained with a primary antibody cocktail containing CD45, EpCAM, biotin-UEA-1, and Ly-51.

Cytometry:

Article Title: Functional variant analyses (FVAs) predict pathogenicity in the BRCA1 DNA double-strand break repair pathway.
Article Snippet: The quantified total p53, phosphop53 and phospho-p53/total p53 ratio were compared for the mutant, VUS and wild-type LCLs. .. Quantitative analysisFlow cytometry was performed using BD FACSAria II equipped with Blue (488 nm), Green (532 nm), Yellow (561 nm), Red (638 nm) and Violet lasers (407 nm). ..

Fluorescence:

Article Title: Kinetics of nuclear uptake and site-specific DNA cleavage during CRISPR-directed gene editing in solid tumor cells
Article Snippet: Lonza program CM-130 was used and after 15 minutes of rest, cells were transferred to a 6-well plate or a 1.5ml tube for further analysis. .. A549 RNP fluorescence was measured by BD FACSAria II (BD Biosciences). .. Cells were harvested by trypsinization, washed three times and resuspended in 1x PBS (−/−) for FACS analysis.

Recombinant:

Article Title: Expansion and CD2/CD3/CD28 stimulation enhance Th2 cytokine secretion of human invariant NKT cells with retained anti-tumor cytotoxicity
Article Snippet: PBMCs were isolated by Ficoll-Paque Plus® density-gradient (GE Healthcare, Piscataway, NJ, USA). .. 2 × 108 PBMCs at 1 × 106 cells/mL were stimulated with 100 ng/mL α-GalCer (KRN7000; Funakoshi, Tokyo, Japan), 100 U/mL recombinant human (rh)IL-2 (Proleukin® , Prometheus, San Diego, CA, USA) and 0.4 ng/mL rhIL-7 (Sigma-Aldrich, St. Louis, MO, USA or R & D Systems, Minneapolis, MN, USA) (or in some expansions 0.4 ng/mL IL-15, R & D Systems, Minneapolis, MN, USA) for 7 days and CD3+Vα24+ iNKT cells sorted using a BD FACSAria-II® (BD Instruments, Santa Clara, CA, USA) to > 95% purity (cell yields > 105 ) or > 90% purity (cell yields between 104 - 105). .. Sorted CD3+ Vα24+ iNKT cells were cultured at 1 × 103 - 5 × 104 cells/mL in T25 or T75 flasks (MidSci, Valley Park, MO, USA) at a 1:50 ratio of iNKT cells to allogeneic irradiated PBMCs (5000 cGy using a 137 Cs source) and stimulated with 50 ng/mL purified NA/LE anti-CD3 (BD Pharmingen, San Jose, CA, USA), adding 100 U/mL rhIL-2 and 0.4 ng/mL or 4 ng/mL rhIL-7 on day 8.

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    Becton Dickinson bd facsaria ii
    Adjuvanticity of FlgE to Soluble Antigen in Mice. ( A–C ) Naïve OVA-specific T cells were purified from the spleens and inguinal lymph nodes of CD45.1 × OTII F1 mice by sorting using various antibodies to surface markers, labeled with eFluor450 and transferred into female C57BL/6 mice at 1 × 10 6 cells/mouse via tail vein injection. Twenty-four hours later, the recipients were treated or immunized via subcutaneous injection at the base of the tail with one of eight compositions, namely 1 μg OVA, 50 μg CpG-1826, 50 μg FlgE, 50 μg FlgEM each alone, or OVA plus the three stimulants individually. Control groups received 100 μL PBS. Three days later, the mice were sacrificed, and the draining inguinal lymph nodes were isolated and photographed ( A ). Single-cell suspension were made for staining with CD45.1 plus CD4 and then run on a BD <t>FACSAria</t> II and analyzed for eFluor450 intensity to quantify the proliferation of OVA-specific T cells ( B,C ). ( D ) To measure the humoral response, WT C57BL/6 mice were immunized with above 8 compositions except for increasing OVA doses to 100 μg. Two weeks later, the mice were sacrificed for serum harvest, and anti-OVA titers were measured using an ELISA as described in Materials and Methods. Data are representatives of two independent experiments that showed similar results. n = 3 mice for panel A–C, and n = 4 mice for panel D. *P
    Bd Facsaria Ii, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Becton Dickinson cd74
    <t>CD74</t> −/− HSPCs show a higher potential to repopulate the BM. Lethally irradiated WT CD45.1 recipient mice were reconstituted with 7.5*10 4 sorted LSK cells from WT (CD45.1), and 7.5 × 10 4 sorted LSK from CD74 −/− (CD45.2) at a 1:1 ratio. Percent of donor-derived cells was analyzed in the BM after 6 and 18 weeks. (A) Total BM cells; Data A in S4 Data (B) myeloid cells; Data B in S4 Data (C) immature BM B cells; Data C in S4 Data and (D) mature BM B cells, Data D in S4 Data . (E) Percent of donor-derived cells was analyzed in LSK and CD34-LSK cells 18 weeks posttransplant. n = 6–8. Data E in S4 Data . n = 6–8. Bars show SEM. Unpaired two-tailed t test * p
    Cd74, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Adjuvanticity of FlgE to Soluble Antigen in Mice. ( A–C ) Naïve OVA-specific T cells were purified from the spleens and inguinal lymph nodes of CD45.1 × OTII F1 mice by sorting using various antibodies to surface markers, labeled with eFluor450 and transferred into female C57BL/6 mice at 1 × 10 6 cells/mouse via tail vein injection. Twenty-four hours later, the recipients were treated or immunized via subcutaneous injection at the base of the tail with one of eight compositions, namely 1 μg OVA, 50 μg CpG-1826, 50 μg FlgE, 50 μg FlgEM each alone, or OVA plus the three stimulants individually. Control groups received 100 μL PBS. Three days later, the mice were sacrificed, and the draining inguinal lymph nodes were isolated and photographed ( A ). Single-cell suspension were made for staining with CD45.1 plus CD4 and then run on a BD FACSAria II and analyzed for eFluor450 intensity to quantify the proliferation of OVA-specific T cells ( B,C ). ( D ) To measure the humoral response, WT C57BL/6 mice were immunized with above 8 compositions except for increasing OVA doses to 100 μg. Two weeks later, the mice were sacrificed for serum harvest, and anti-OVA titers were measured using an ELISA as described in Materials and Methods. Data are representatives of two independent experiments that showed similar results. n = 3 mice for panel A–C, and n = 4 mice for panel D. *P

    Journal: Scientific Reports

    Article Title: Flagellar Hooks and Hook Protein FlgE Participate in Host Microbe Interactions at Immunological Level

    doi: 10.1038/s41598-017-01619-1

    Figure Lengend Snippet: Adjuvanticity of FlgE to Soluble Antigen in Mice. ( A–C ) Naïve OVA-specific T cells were purified from the spleens and inguinal lymph nodes of CD45.1 × OTII F1 mice by sorting using various antibodies to surface markers, labeled with eFluor450 and transferred into female C57BL/6 mice at 1 × 10 6 cells/mouse via tail vein injection. Twenty-four hours later, the recipients were treated or immunized via subcutaneous injection at the base of the tail with one of eight compositions, namely 1 μg OVA, 50 μg CpG-1826, 50 μg FlgE, 50 μg FlgEM each alone, or OVA plus the three stimulants individually. Control groups received 100 μL PBS. Three days later, the mice were sacrificed, and the draining inguinal lymph nodes were isolated and photographed ( A ). Single-cell suspension were made for staining with CD45.1 plus CD4 and then run on a BD FACSAria II and analyzed for eFluor450 intensity to quantify the proliferation of OVA-specific T cells ( B,C ). ( D ) To measure the humoral response, WT C57BL/6 mice were immunized with above 8 compositions except for increasing OVA doses to 100 μg. Two weeks later, the mice were sacrificed for serum harvest, and anti-OVA titers were measured using an ELISA as described in Materials and Methods. Data are representatives of two independent experiments that showed similar results. n = 3 mice for panel A–C, and n = 4 mice for panel D. *P

    Article Snippet: Cells were run on a BD FACSAria II and analyzed for eFluor450 intensity to reflect proliferation.

    Techniques: Mouse Assay, Purification, Labeling, Injection, Isolation, Staining, Enzyme-linked Immunosorbent Assay

    Higher-Order Clustering is Inhibited by the Unliganded Extracellular Domain ( A ) Schematic illustration of possible mechanism underlying proteolytic activation of DR5. ( B )Comparing TRAIL sensitivity of the DR5-TEV-expressing BJAB cells to that of the DR5-WT-expressing BJAB cells. Caspase-8 activity was measured using the CaspGLOW red Caspase-8 activity kit (Biovision) and flow cytometry (BD FACSAria II) and was calculated as percentage of DsRed2+/GFP+ cells divided by percentage of GFP+ cells. Results were from 3 independent experiments (n=3), and expressed as mean ± SEM. ( C )TEV-induced apoptosis. DR5-deficient BJAB cells were transiently transfected with WT DR5-EGFP with (DR5-TEV) or without (DR5-WT) TEV cleavage site between the ECD and TMD. After transfection, BJAB cells were treated with TEV for 5 h before analysis. ( D )Examples of CFP and YFP photobleaching for WT DR5 without (left) and with (right) TRAIL. ( E )Receptor self-association of the WT DR5 and mutants. The effects of dimer-breaking mutation (G217Y) and trimer-breaking mutation (A222Y) with or without TRAIL pre-treatment are quantitated in the form of FRET efficiency. CFP/YFP co-transfected cells were used as FRET negative control. N.S indicates not significant. Twelve regions of interest (n=12) (ROI, e.g., plasma membrane bound DR5) from different cells were examined by FRET study. .

    Journal: Cell

    Article Title: Higher-Order Clustering of the Transmembrane Anchor of DR5 Drives Signaling

    doi: 10.1016/j.cell.2019.02.001

    Figure Lengend Snippet: Higher-Order Clustering is Inhibited by the Unliganded Extracellular Domain ( A ) Schematic illustration of possible mechanism underlying proteolytic activation of DR5. ( B )Comparing TRAIL sensitivity of the DR5-TEV-expressing BJAB cells to that of the DR5-WT-expressing BJAB cells. Caspase-8 activity was measured using the CaspGLOW red Caspase-8 activity kit (Biovision) and flow cytometry (BD FACSAria II) and was calculated as percentage of DsRed2+/GFP+ cells divided by percentage of GFP+ cells. Results were from 3 independent experiments (n=3), and expressed as mean ± SEM. ( C )TEV-induced apoptosis. DR5-deficient BJAB cells were transiently transfected with WT DR5-EGFP with (DR5-TEV) or without (DR5-WT) TEV cleavage site between the ECD and TMD. After transfection, BJAB cells were treated with TEV for 5 h before analysis. ( D )Examples of CFP and YFP photobleaching for WT DR5 without (left) and with (right) TRAIL. ( E )Receptor self-association of the WT DR5 and mutants. The effects of dimer-breaking mutation (G217Y) and trimer-breaking mutation (A222Y) with or without TRAIL pre-treatment are quantitated in the form of FRET efficiency. CFP/YFP co-transfected cells were used as FRET negative control. N.S indicates not significant. Twelve regions of interest (n=12) (ROI, e.g., plasma membrane bound DR5) from different cells were examined by FRET study. .

    Article Snippet: Flow cytometry was performed on BD FACSAria II.

    Techniques: Activation Assay, Expressing, Activity Assay, Flow Cytometry, Transfection, Mutagenesis, Negative Control

    CD74 −/− HSPCs show a higher potential to repopulate the BM. Lethally irradiated WT CD45.1 recipient mice were reconstituted with 7.5*10 4 sorted LSK cells from WT (CD45.1), and 7.5 × 10 4 sorted LSK from CD74 −/− (CD45.2) at a 1:1 ratio. Percent of donor-derived cells was analyzed in the BM after 6 and 18 weeks. (A) Total BM cells; Data A in S4 Data (B) myeloid cells; Data B in S4 Data (C) immature BM B cells; Data C in S4 Data and (D) mature BM B cells, Data D in S4 Data . (E) Percent of donor-derived cells was analyzed in LSK and CD34-LSK cells 18 weeks posttransplant. n = 6–8. Data E in S4 Data . n = 6–8. Bars show SEM. Unpaired two-tailed t test * p

    Journal: PLoS Biology

    Article Title: CD74 is a regulator of hematopoietic stem cell maintenance

    doi: 10.1371/journal.pbio.3001121

    Figure Lengend Snippet: CD74 −/− HSPCs show a higher potential to repopulate the BM. Lethally irradiated WT CD45.1 recipient mice were reconstituted with 7.5*10 4 sorted LSK cells from WT (CD45.1), and 7.5 × 10 4 sorted LSK from CD74 −/− (CD45.2) at a 1:1 ratio. Percent of donor-derived cells was analyzed in the BM after 6 and 18 weeks. (A) Total BM cells; Data A in S4 Data (B) myeloid cells; Data B in S4 Data (C) immature BM B cells; Data C in S4 Data and (D) mature BM B cells, Data D in S4 Data . (E) Percent of donor-derived cells was analyzed in LSK and CD34-LSK cells 18 weeks posttransplant. n = 6–8. Data E in S4 Data . n = 6–8. Bars show SEM. Unpaired two-tailed t test * p

    Article Snippet: Generation of chimeric mice For the microenvironment experiment: Lethally irradiated (950 Rad) C57BL/6 (WT) recipient mice were reconstituted with 5*106 WT or CD74−/− BM cells.

    Techniques: Irradiation, Mouse Assay, Derivative Assay, Two Tailed Test

    CD74 −/− HSPCs demonstrate enhanced long-term self-renewal capacity. (A–F) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 3:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 3:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (A) Total BM cells; Data A in S5 Data (B) BM myeloid cells; Data B in S5 Data (C) BM immature B cells; Data C in S5 Data (D) LSK; Data D in S5 Data (E) CD34-/LSK; Data E in S5 Data (F) Mature BM B cells; Data F in S5 Data . n = 13. (G–L) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 9:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 9:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor-derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (G) Total BM cells; Data G in S5 Data (H) BM myeloid cells; Data H in S5 Data (I) BM immature B cells; Data I in S5 Data (J) LSK; Data J in S5 Data (K) CD34-/LSK; Data K in S5 Data (L) Mature BM B cells; Data L in S5 Data . n = 12. Bars show SEM. Unpaired two-tailed t test * p

    Journal: PLoS Biology

    Article Title: CD74 is a regulator of hematopoietic stem cell maintenance

    doi: 10.1371/journal.pbio.3001121

    Figure Lengend Snippet: CD74 −/− HSPCs demonstrate enhanced long-term self-renewal capacity. (A–F) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 3:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 3:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (A) Total BM cells; Data A in S5 Data (B) BM myeloid cells; Data B in S5 Data (C) BM immature B cells; Data C in S5 Data (D) LSK; Data D in S5 Data (E) CD34-/LSK; Data E in S5 Data (F) Mature BM B cells; Data F in S5 Data . n = 13. (G–L) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 9:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 9:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor-derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (G) Total BM cells; Data G in S5 Data (H) BM myeloid cells; Data H in S5 Data (I) BM immature B cells; Data I in S5 Data (J) LSK; Data J in S5 Data (K) CD34-/LSK; Data K in S5 Data (L) Mature BM B cells; Data L in S5 Data . n = 12. Bars show SEM. Unpaired two-tailed t test * p

    Article Snippet: Generation of chimeric mice For the microenvironment experiment: Lethally irradiated (950 Rad) C57BL/6 (WT) recipient mice were reconstituted with 5*106 WT or CD74−/− BM cells.

    Techniques: Irradiation, Mouse Assay, Derivative Assay, Transplantation Assay, Two Tailed Test

    Expansion of HSPCs in the BM of CD74 −/− mice. (A) BM cells derived from WT or CD74 −/− were purified. Histograms show representative analysis of CD74 expression on HSPCs in WT and CD74 −/− mice. n = 3. (B) Total BM cellularity per femur and tibia in WT and CD74 −/− mice, Data A in S1 Data . (C–J) The percent of the different populations in WT and CD74 −/− -derived BM cells. (C) Lin-; Data B in S1 Data (D) Representative FACS analysis of WT and CD74 −/− HSPCs; (E) LSK; Data C in S1 Data (F) CD34-/LSK; Data D in S1 Data and (G) CD34+; Data E in S1 Data (H) CD150+CD48-LSK; Data F in S1 Data (I) CD150-CD48-/LSK; Data G in S1 Data and (J) CD150-CD48+/LSK; n = 14–18, Data H in S1 Data . (K) CFUC assay: Total BM cells from WT and CD74 −/− mice were seeded at 15,000 cells/mL in semisolid cultures supplemented with cytokines and nutrients. CFU-C were counted 7 days later; n = 7, Data I in S1 Data . Bars show SEM. Unpaired two-tailed t test * p

    Journal: PLoS Biology

    Article Title: CD74 is a regulator of hematopoietic stem cell maintenance

    doi: 10.1371/journal.pbio.3001121

    Figure Lengend Snippet: Expansion of HSPCs in the BM of CD74 −/− mice. (A) BM cells derived from WT or CD74 −/− were purified. Histograms show representative analysis of CD74 expression on HSPCs in WT and CD74 −/− mice. n = 3. (B) Total BM cellularity per femur and tibia in WT and CD74 −/− mice, Data A in S1 Data . (C–J) The percent of the different populations in WT and CD74 −/− -derived BM cells. (C) Lin-; Data B in S1 Data (D) Representative FACS analysis of WT and CD74 −/− HSPCs; (E) LSK; Data C in S1 Data (F) CD34-/LSK; Data D in S1 Data and (G) CD34+; Data E in S1 Data (H) CD150+CD48-LSK; Data F in S1 Data (I) CD150-CD48-/LSK; Data G in S1 Data and (J) CD150-CD48+/LSK; n = 14–18, Data H in S1 Data . (K) CFUC assay: Total BM cells from WT and CD74 −/− mice were seeded at 15,000 cells/mL in semisolid cultures supplemented with cytokines and nutrients. CFU-C were counted 7 days later; n = 7, Data I in S1 Data . Bars show SEM. Unpaired two-tailed t test * p

    Article Snippet: Generation of chimeric mice For the microenvironment experiment: Lethally irradiated (950 Rad) C57BL/6 (WT) recipient mice were reconstituted with 5*106 WT or CD74−/− BM cells.

    Techniques: Mouse Assay, Derivative Assay, Purification, Expressing, FACS, Two Tailed Test

    CD74 −/− HSPCs have an advantage in BM repopulation. Lethally irradiated WT (CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT (CD45.2) at a 1:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 1:1 ratio. (A) Representative BM FACS staining. Percent of donor-derived cells was analyzed in the BM after 6, 16, and 24 weeks in (B) Total BM cells; Data A in S3 Data (C) myeloid cells (CD11B+); Data B in S3 Data (D) B cells (B220+); Data C in S3 Data (E) LSK; Data D in S3 Data (F) CD34-/LSK; Data E in S3 Data (G) immature BM B cells (B220+IgD-); Data F in S3 Data (H) mature BM B cells (B220+ IgM+ IgD+); Data G in S3 Data . n = 8–18. Bars show SEM. Unpaired two-tailed t test * p

    Journal: PLoS Biology

    Article Title: CD74 is a regulator of hematopoietic stem cell maintenance

    doi: 10.1371/journal.pbio.3001121

    Figure Lengend Snippet: CD74 −/− HSPCs have an advantage in BM repopulation. Lethally irradiated WT (CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT (CD45.2) at a 1:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 1:1 ratio. (A) Representative BM FACS staining. Percent of donor-derived cells was analyzed in the BM after 6, 16, and 24 weeks in (B) Total BM cells; Data A in S3 Data (C) myeloid cells (CD11B+); Data B in S3 Data (D) B cells (B220+); Data C in S3 Data (E) LSK; Data D in S3 Data (F) CD34-/LSK; Data E in S3 Data (G) immature BM B cells (B220+IgD-); Data F in S3 Data (H) mature BM B cells (B220+ IgM+ IgD+); Data G in S3 Data . n = 8–18. Bars show SEM. Unpaired two-tailed t test * p

    Article Snippet: Generation of chimeric mice For the microenvironment experiment: Lethally irradiated (950 Rad) C57BL/6 (WT) recipient mice were reconstituted with 5*106 WT or CD74−/− BM cells.

    Techniques: Irradiation, Mouse Assay, Derivative Assay, FACS, Staining, Two Tailed Test

    CD74 can serve as a potential target for therapy. (A, B) WT and CD74 −/− BM cells were cultured alone or incubated with blocking anti-CD74 antibody (20, 50, and 100 μg/ml). After 48 h, percent LSK from live cells was analyzed by FACS; n = 4–7, Data A and B in S8 Data . (C, D) Survival curve: 5-FU (150 mg/kg and 125mg/kg) was injected to WT and CD74 −/− mice once a week. Log-rank test *

    Journal: PLoS Biology

    Article Title: CD74 is a regulator of hematopoietic stem cell maintenance

    doi: 10.1371/journal.pbio.3001121

    Figure Lengend Snippet: CD74 can serve as a potential target for therapy. (A, B) WT and CD74 −/− BM cells were cultured alone or incubated with blocking anti-CD74 antibody (20, 50, and 100 μg/ml). After 48 h, percent LSK from live cells was analyzed by FACS; n = 4–7, Data A and B in S8 Data . (C, D) Survival curve: 5-FU (150 mg/kg and 125mg/kg) was injected to WT and CD74 −/− mice once a week. Log-rank test *

    Article Snippet: Generation of chimeric mice For the microenvironment experiment: Lethally irradiated (950 Rad) C57BL/6 (WT) recipient mice were reconstituted with 5*106 WT or CD74−/− BM cells.

    Techniques: Cell Culture, Incubation, Blocking Assay, FACS, Injection, Mouse Assay

    Accumulation of HSPCs is not CXCR4 dependent. (A, B) FACS analysis of CXCR4 expression on BM LSK and BM CD34-/LSK of WT and CD74 −/− mice, n = 7, Data A in S6 Data . Representative histograms are shown. (C–E) FACS analysis for HSPCs in the PB of WT and CD74 −/− . (C) Dot plot analysis of LSK in WT and CD74 −/− mice. (D, E) Cell number of (D) LSK and (E) CD34-LSK in 600 μl blood. WT n = 6 CD74 −/− n = 7, Data B and C in S6 Data . (F) AMD3100 (20 mg/kg −1 ) was injected to WT and CD74 −/− mice. After 2 h, percent of LSK in the PB was analyzed; n = 9–11, Data D in S6 Data . (G–I) FACS staining of WT and CD74 −/− HSPCs for Ki-67. Results are presented as: (G) percent of CD34-/LSK Ki-67 and CD34-/LSK Ki-67+ from total BM cells, Data E in S6 Data ; (H) percent of CD34+/LSK Ki-67- and CD34+/LSK Ki-67+ from total BM cells, Data F in S6 Data ; and (I) percent of Ki-67+ from CD34-/LSK and percent of Ki-67+ from CD34+LSK, n = 15, Data G in S6 Data . (J, K) Mice were fed with 0.8 mg/ml BrdU in their drinking water for 3 days, and BrdU incorporation was analyzed by FACS. Results are represented as: (J) percent of LSK BrdU- and LSK BrdU+ from total BM cells, Data H in S6 Data ; (K) percent of BrdU+ in LSK; n = 12–14, Data I in S6 Data . Bars show SEM. Unpaired two-tailed t test: * p

    Journal: PLoS Biology

    Article Title: CD74 is a regulator of hematopoietic stem cell maintenance

    doi: 10.1371/journal.pbio.3001121

    Figure Lengend Snippet: Accumulation of HSPCs is not CXCR4 dependent. (A, B) FACS analysis of CXCR4 expression on BM LSK and BM CD34-/LSK of WT and CD74 −/− mice, n = 7, Data A in S6 Data . Representative histograms are shown. (C–E) FACS analysis for HSPCs in the PB of WT and CD74 −/− . (C) Dot plot analysis of LSK in WT and CD74 −/− mice. (D, E) Cell number of (D) LSK and (E) CD34-LSK in 600 μl blood. WT n = 6 CD74 −/− n = 7, Data B and C in S6 Data . (F) AMD3100 (20 mg/kg −1 ) was injected to WT and CD74 −/− mice. After 2 h, percent of LSK in the PB was analyzed; n = 9–11, Data D in S6 Data . (G–I) FACS staining of WT and CD74 −/− HSPCs for Ki-67. Results are presented as: (G) percent of CD34-/LSK Ki-67 and CD34-/LSK Ki-67+ from total BM cells, Data E in S6 Data ; (H) percent of CD34+/LSK Ki-67- and CD34+/LSK Ki-67+ from total BM cells, Data F in S6 Data ; and (I) percent of Ki-67+ from CD34-/LSK and percent of Ki-67+ from CD34+LSK, n = 15, Data G in S6 Data . (J, K) Mice were fed with 0.8 mg/ml BrdU in their drinking water for 3 days, and BrdU incorporation was analyzed by FACS. Results are represented as: (J) percent of LSK BrdU- and LSK BrdU+ from total BM cells, Data H in S6 Data ; (K) percent of BrdU+ in LSK; n = 12–14, Data I in S6 Data . Bars show SEM. Unpaired two-tailed t test: * p

    Article Snippet: Generation of chimeric mice For the microenvironment experiment: Lethally irradiated (950 Rad) C57BL/6 (WT) recipient mice were reconstituted with 5*106 WT or CD74−/− BM cells.

    Techniques: FACS, Expressing, Mouse Assay, Injection, Staining, BrdU Incorporation Assay, Two Tailed Test

    CD74 −/− HSPC expansion is cell intrinsic. Lethally irradiated WT or CD74 −/− mice were transplanted with either WT or CD74 −/− total BM cells. Long-term reconstitution was evaluated 16 weeks posttransplantation. Percent of total BM cells was calculated for (A) LIN -; Data A in S2 Data (B) LSK; Data B in S2 Data and (C) CD34-/LSK; Data C in S2 Data n = 5–12. Bars show SEM. Unpaired two-tailed t test *

    Journal: PLoS Biology

    Article Title: CD74 is a regulator of hematopoietic stem cell maintenance

    doi: 10.1371/journal.pbio.3001121

    Figure Lengend Snippet: CD74 −/− HSPC expansion is cell intrinsic. Lethally irradiated WT or CD74 −/− mice were transplanted with either WT or CD74 −/− total BM cells. Long-term reconstitution was evaluated 16 weeks posttransplantation. Percent of total BM cells was calculated for (A) LIN -; Data A in S2 Data (B) LSK; Data B in S2 Data and (C) CD34-/LSK; Data C in S2 Data n = 5–12. Bars show SEM. Unpaired two-tailed t test *

    Article Snippet: Generation of chimeric mice For the microenvironment experiment: Lethally irradiated (950 Rad) C57BL/6 (WT) recipient mice were reconstituted with 5*106 WT or CD74−/− BM cells.

    Techniques: Irradiation, Mouse Assay, Two Tailed Test

    CD74 regulates the survival of HSPCs and CD18 expression. (A, B) FACS staining of WT and CD74-deficient HSPCs for ROS. (A) Results are presented as the number of ROS high cells per 10 6 cells, n = 9, Data A in S7 Data . (B) Percentage of ROS high in LSK, Data B in S7 Data . (C, D) Percent of LSK (C) Data C in S7 Data , and CD34- (D) after 6 days of NAC injections (50 mg kg −1 ); n = 5, Data D in S7 Data . (E, F) FACS analysis of HSPCs from WT and CD74 −/− mice for Annexin V (E); n = 10–12, Data E in S7 Data , and after 24 h under hypoxic (F); n = 3 (each dot represents a duplicate determination), Data F in S7 Data . (G) Ratio of Annexin V+ CD74 −/− to WT of HSPCs under hypoxic and normoxic conditions, Data G in S7 Data . (H, I) FACS analysis of HSCs from WT and CD74 −/− mice for HIF-1α; n = 7–8, Data H in S7 Data . (J) Sorted WT and CD74 −/− CD34-/LSK cells were analyzed for CD18 mRNA levels; n = 3. The bars show the DESeq2 normalized counts for the CD18 gene, Data I in S7 Data . (K) Binding of CD74–ICD to CD18 promoter and intron regions in Lin− samples. ChIP-seq analysis using anti-CD74 antibody. (L) FACS analysis of HSCs from WT and CD74 −/− mice for CD18. Graph summarizes the results of 6 mice in each group, Data J in S7 Data . (M) FACS analysis of HSCs from WT and MIF −/− mice for CD18; n = 3, Data K in S7 Data . (N) WT and CD74 −/− BM were cultured with or without the MIF inhibitor, ISO-1, for 48 h, percent CD18 on CD34-/LSK was analyzed by FACS; n = 6, Data L in S7 Data . (O) WT (CD45.1) Lin negative cells were cultured in the presence of WT (CD45.2) total BM or MIF −/− (CD45.2) total BM for 48 h. The percent CD18 on CD34-/LSK cells (CD45.1) was analyzed by FACS; n = 8, Data M in S7 Data . Bars show SEM. Unpaired two-tailed t test * p

    Journal: PLoS Biology

    Article Title: CD74 is a regulator of hematopoietic stem cell maintenance

    doi: 10.1371/journal.pbio.3001121

    Figure Lengend Snippet: CD74 regulates the survival of HSPCs and CD18 expression. (A, B) FACS staining of WT and CD74-deficient HSPCs for ROS. (A) Results are presented as the number of ROS high cells per 10 6 cells, n = 9, Data A in S7 Data . (B) Percentage of ROS high in LSK, Data B in S7 Data . (C, D) Percent of LSK (C) Data C in S7 Data , and CD34- (D) after 6 days of NAC injections (50 mg kg −1 ); n = 5, Data D in S7 Data . (E, F) FACS analysis of HSPCs from WT and CD74 −/− mice for Annexin V (E); n = 10–12, Data E in S7 Data , and after 24 h under hypoxic (F); n = 3 (each dot represents a duplicate determination), Data F in S7 Data . (G) Ratio of Annexin V+ CD74 −/− to WT of HSPCs under hypoxic and normoxic conditions, Data G in S7 Data . (H, I) FACS analysis of HSCs from WT and CD74 −/− mice for HIF-1α; n = 7–8, Data H in S7 Data . (J) Sorted WT and CD74 −/− CD34-/LSK cells were analyzed for CD18 mRNA levels; n = 3. The bars show the DESeq2 normalized counts for the CD18 gene, Data I in S7 Data . (K) Binding of CD74–ICD to CD18 promoter and intron regions in Lin− samples. ChIP-seq analysis using anti-CD74 antibody. (L) FACS analysis of HSCs from WT and CD74 −/− mice for CD18. Graph summarizes the results of 6 mice in each group, Data J in S7 Data . (M) FACS analysis of HSCs from WT and MIF −/− mice for CD18; n = 3, Data K in S7 Data . (N) WT and CD74 −/− BM were cultured with or without the MIF inhibitor, ISO-1, for 48 h, percent CD18 on CD34-/LSK was analyzed by FACS; n = 6, Data L in S7 Data . (O) WT (CD45.1) Lin negative cells were cultured in the presence of WT (CD45.2) total BM or MIF −/− (CD45.2) total BM for 48 h. The percent CD18 on CD34-/LSK cells (CD45.1) was analyzed by FACS; n = 8, Data M in S7 Data . Bars show SEM. Unpaired two-tailed t test * p

    Article Snippet: Generation of chimeric mice For the microenvironment experiment: Lethally irradiated (950 Rad) C57BL/6 (WT) recipient mice were reconstituted with 5*106 WT or CD74−/− BM cells.

    Techniques: Expressing, FACS, Staining, Mouse Assay, Binding Assay, Chromatin Immunoprecipitation, Cell Culture, Two Tailed Test