MHCD0417

antibody from Invitrogen Antibodies

Targeting: CD4

Provider product page for MHCD0417

Flow cytometry

Other assay

Antibody data

Antibody Data
Antigen structure
References [26]
Comments [0]
Validations
Flow cytometry [1]
Other assay [22]

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Product number: MHCD0417 - Provider product page
Provider: Invitrogen Antibodies
Product name: CD4 Monoclonal Antibody (S3.5), PE-Texas Red
Antibody type: Monoclonal
Antigen: Other
Description: The R-phycoerythrin (PE)-Texas Red conjugate permits simultaneous multicolor labeling and detection of multiple targets with excitation by a single excitation source-the 488 nm line of the argon-ion laser.
Reactivity: Human
Host: Mouse
Isotype: IgG
Antibody clone number: S3.5
Vial size: 500 µL
Storage: 4° C, store in dark

CD4 protein structure - MHCD0417 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 1 T H 17 and T Reg cells are enriched in the healthy human cecum. A) Representative gating strategy for the FACS analysis of lamina propria mononuclear cells from pinch biopsies obtained from four regions of healthy colonic mucosa. B) Representative FACS plots for two subjects (CAP9 and CAP24) showing IL-17 + cells (gated on CD4 + cells) from four biopsy locations in each subject. C) Cumulative data of IL-17 + CD4 + cells for all subjects. The top panel includes data from samples from all subjects with available flow cytometry data (N = 19) whereas the bottom panel shows data from subjects who have a complete set of paired samples from all four biopsy locations (N = 15). D) Representative FACS plots for two subjects (CAP9 and CAP23) showing CD25 + FoxP3 + cells (gated on CD4 + cells). E) Cumulative data of CD25 + FoxP3 + cells for all subjects. F) T H 17/T Reg ratios were plotted for all subjects according to biopsy location. Unless otherwise indicated, differences were not significant. * P

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 4 T H 1 and T H 2 lymphocyte populations do not vary by region. A) Representative gating strategy showing IFNgamma and IL-4 staining of CD4 + cells isolated from four regions of the intestine in two subjects (CAP 20 and CAP23). B) IFNgamma and C) IL-4 production by CD4 + isolated from each region for all subjects. Differences between regions are not statistically significant (two tailed Mann-Whitney). TI: Terminal Ileum, ICV: Ileocecal Valve, AO: Appendiceal Orifice, SC: Sigmoid Colon.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 3 T H 22 and IL-17 + FoxP3 + cells are enriched in the healthy human cecum. A) Representative FACS plots for two subjects (CAP9 and CAP12) showing intracellular cytokine staining for IL-17 and IL-22 of lamina propria CD4 + cells. B) Cumulative data of IL-22 + IL-17 - CD4 + cells for all subjects. The top panel includes data from all samples from all subjects (N = 19) whereas the bottom panel shows data from subjects (N = 15) who have a complete set of paired samples from all four biopsy locations. C) Representative FACS plots for two subjects (CAP23 and CAP26) showing staining for IL-17 + FoxP3 + lamina propria CD4 + cells. D) Cumulative data of IL-17 + FoxP3 + CD4 + cells for all subjects. Unless otherwise indicated, differences were not significant. * P

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 5 Polycytokine producing CD4 + cells analyzed by Boolean gate analysis from different regions of the intestinal tract. A) Boolean gates on IL-17, IL-22, IL-4, TNFalpha and IFNgamma positive lamina propria CD4 + cells. Shown are representative FACS plots gated on CD4 + cells from the ileocecal valve of two subjects (CAP9 and CAP10). B) Pie charts showing the averaged pattern of cytokine production in CD4 + cells from the different regions of the gut (from N = 22 subjects). Each slice within the pie chart represents a specific combination of cytokine staining (denoted in C). Data is shown only for combinations with frequencies >2% of the total CD4 + population. IL-22 + TNFalpha + IFNgamma + cells are >2% of CD4 + cells only from the ileocecal valve and appendiceal orifice. The permutation test at 10,000 permutations did not demonstrate significant differences among the pie charts represented.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 2. Isolation and epitope mapping of H1-HA-reactive CD4 + T cells from naive and memory compartments of influenza vaccinated donors. (A) Naive and memory CD4 + T cells were FACS sorted from PBMCs of four donors and polyclonally expanded in multiple wells, each containing 1,000-2,000 cells. The number of wells ranged from 150 to 384, depending on the number of cells isolated (see Materials and methods). After 14-21 d, the amplified naive and memory T cell libraries were screened against a pool of overlapping peptides spanning the entire H1-HA sequence in the presence of autologous APCs. Proliferation was assessed on day 4 after a 16-h pulse with [ 3 H]thymidine. Data are expressed as the counts per minute, after subtraction of background proliferation (Deltacpm). The proliferative response of each T cell clone in the library is represented by a single dot. The specificity of positive cultures was confirmed in three independent experiments. (B) Frequencies of H1-HA-specific T cells within naive or memory CD4 + T cells was calculated based on number of negative wells according to the Poisson distribution. Data are expressed as frequency per million of naive or memory CD4 + T cells. Each symbol indicates a different donor. (C) Epitope mapping of H1-HA-specific T cell clones from naive or memory libraries. The epitopes were identified by screening the T cell cultures with overlapping peptides spanning the entire H1-HA sequence in at least two independent experiments, with co

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 3. H1-HA-specific naive and memory CD4 + T cell clones show different functional avidities. Functional avidity of H1-HA-reactive CD4 + T cell clones isolated from donor HD1 was determined by stimulation with titrated doses of synthetic H1-HA peptides or recombinant H1-HA in the presence of autologous monocytes. Proliferation was assessed on day 3 after a 16-h pulse with [ 3 H]thymidine; EC 50 values were calculated by nonlinear regression curve fit. Shown are mean data of one experiment done with each clone in duplicate and are representative of three independent experiments. (A) Violin plots of the frequency distribution of reciprocal EC 50 values of T cell clones from the memory or naive compartment stimulated with titrated doses of H1-HA peptides. T cell clones specific for the immunodominant H1-HA 411-430 epitope are reported as red dots. Lines represent the median and quartiles. ***, P < 0.001 as determined by two-tailed Mann-Whitney U test. (B and C) Scatter plots of reciprocal EC 50 values of T cell clones from the memory (B) or naive (C) compartment, stimulated in parallel with recombinant H1-HA (x axis) and synthetic peptides (y axis). EC 50 values below the detection limit for stimulations with recombinant H1-HA were set arbitrarily to 20 ug/ml; the corresponding T cell clones are reported as white dots. Spearman correlation was calculated based on EC 50 pairs from T cell clones responding to both peptides and recombinant H1-HA (B, n = 36; C, n = 29). Thresho

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 5 Cross-reactive T cells to Enterobacteriaceae are already detected in the naive repertoire. (A) Human naive CD4 + CD45RA + CD95 - T cells were flow cytometry-sorted at high purity, labeled with CFSE and primed in vitro for 10 days with the indicated heat-inactivated bacteria and autologous monocytes in absence (upper panel) or presence (lower panel) of anti-MHC-II blocking antibodies. Shown are the CFSE profiles by flow cytometry on day 10 after priming and the percentages of CFSE low proliferating cells from a representative donor out of a total of 6 different donors tested in 6 independent experiments. (B, C) Antigen-primed CFSE low T cells were cloned by limiting dilution. T cell clones isolated from each CFSE low fraction were screened with a panel of heat-inactivated bacteria in the presence of autologous monocytes. Proliferation was assessed on day 3 after a 16-h pulse with [ 3 H]-thymidine and expressed as counts per min (Cpm). The heatmaps report the Cpm of T cell clones isolated from CFSE low fractions of a representative donor. The data are representative of at least two independent experiments (B). The antigen used for the initial in vitro priming and the number of clones tested are reported on top of each heatmap. Each row of the heatmaps refers to an individual T cell clone, stimulated with the antigens reported at the bottom. (C) Pooled data of the patterns of reactivity of T cell clones isolated from each CFSE low fraction of in vitro primed naive CD4 +

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 3 Memory CD4 + T cells are broadly cross-reactive to Enterobacteriaceae. Bacteria-reactive polyclonal CD4 + T cell lines were obtained by flow cytometry-sorting of CFSE low cells from primary cultures of memory CD4 + T cells. Following expansion, the cells were re-labeled with CFSE and restimulated with the same bacteria (homologous restimulation) or with different bacteria (heterologous restimulation) in the presence of autologous monocytes and measured by flow cytometry. (A) CFSE profiles of polyclonal T cell lines from a representative donor reactive for the bacteria indicated on top and restimulated for 5 days with the bacteria indicated on the right. Homologous restimulation for each culture is highlighted with a grey shadow. (B) Percentage of CFSE low cells after secondary homologous or heterologous restimulation in n = 5 different donors (mean values +- SEM) in five independent experiments. CFSE low cells are reported as percentage of total events after normalization on live lymphocytes. * p -value < 0.05, ** p -value < 0.01, as determined by two tailed paired t -test compared to positive control (homologous antigen in the re-stimulation). (C and D) CFSE low cells from primary cultures of memory CD4 + T cells were also used to isolate a large panel of Enterobacteriaceae-reactive T cell clones by limiting dilution. The T cell clones specific for the bacteria indicated on top of the heatmaps in C were then screened for cross-reactivity with a panel of bacteria as

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 4 Extensive clonotype sharing among Enterobacteriaceae-reactive memory CD4 + T cell repertoires. Bacteria-reactive CFSE low cells were flow cytometry-sorted from stimulated memory CD4 + T cells, and their TCR Vbeta repertoire was determined by deep sequencing. (A) Number of unique productive TCR Vbeta nucleotide rearrangements resolved from each bacteria-responsive memory repertoire in n = 2-4 different donors in two independent experiments. Each symbol represents a different donor. Bars indicate mean +- SD ( n = 2-4). (B) Pairwise similarity of each antigen-reactive TCR Vbeta repertoire was calculated by Chao-Jaccard overlap. The reciprocal distance of each TCR Vbeta repertoire was plotted using Kruskal''s nonmetric multidimensional scaling. Each point of the graph represents a distinct bacteria-reactive memory CD4 + TCR Vbeta repertoire (average from n = 2-4 donors): the closer the points, the more similar the repertoires in terms of sharing of TCR Vbeta clonotypes. (C) Violin plots of the frequency distribution of TCR Vbeta clonotypes reactive to Enterobacteriaceae or control bacteria from a representative donor. Each dot represents a unique TCR Vbeta nucleotide rearrangement, and the color code indicates the number of distinct Enterobacteriaceae-reactive repertoires in which the clonotype was found. Median values are reported as dashed lines. The number of clonotypes for each class of reactivity is reported on top. Dotted lines in the graphs indicate the frequency

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 1 Enterobacteriaceae-reactive memory CD4 + T cells are present in the blood of healthy donors and show a Th1*/17 phenotype. Human memory CD4 + T cells were isolated from PBMCs, labeled with CFSE and stimulated with the indicated heat-inactivated bacteria in the presence of autologous monocytes and analysed by flow cytometry. (A) CFSE profiles on day 6 in a representative donor. Numbers indicate percentage of CFSE low proliferating T cells. (B) Percentages of CFSE low cells in n =17-40 different donors analyzed in 20 independent experiments (1 or 2 donors per experiment). Each dot represents a donor, boxes are quartile values, whiskers represent the highest and lowest values, and lines represent the median values. Numbers on top indicate the total number of donors analyzed with the indicated bacteria **** p -value < 0.0001, as determined by two-tailed unpaired t -test. (C and D) IFN-gamma, IL-17A, and IL-22 production by CFSE low cells was measured by intracellular staining after stimulation with PMA and ionomycin in the presence of Brefeldin A in a representative donor (C) and in n = 4-14 different donors in seven independent experiments (D). In (C), numbers in quadrants indicate percent cells in each throughout. In (D), each dot represents a donor, boxes are quartile values, whiskers represent the highest and lowest values, and lines represent the median values. Shown are total percentages of cells producing IFN-gamma, IL-17, or IL-22, irrespective of the fact that th

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 2 Septic patients with systemic K. pneumoniae infection show reduced frequency of circulating CXCR3 + T cells and selectively lack K. pneumoniae -reactive T cells. (A) The surface expression of chemokine receptors by memory CD4 + T cells from PBMCs of n = 9 healthy controls and n = 6 septic patients with systemic K. pneumoniae infection was analyzed by flow cytometry. Shown are pooled data (mean +- SD, five independent experiments with samples from one or two septic patients and one or two healthy controls per experiment) of percentage of memory CD4 + T cells positive for combinations of chemokine receptors. (B) Human memory CCR6 + CXCR3 + CCR4 - Th cells were sorted, labeled with CFSE, and stimulated with the indicated antigens in the presence of autologous monocytes. Shown are CFSE profiles on day 6 in a representative septic patient (upper panel), and pooled data of percentage of CFSE low cells in healthy controls or septic patients (lower panel). Each dot represents a donor, boxes are quartile values, whiskers represent highest and lowest values, and lines represent the median values. ** p -value < 0.01, *** p -value < 0.001, as determined by two-tailed unpaired t -test. The data are from three independent experiments with samples from one or two septic patients and two healthy controls per experiment.