25-0048-42

antibody from Invitrogen Antibodies

Targeting: CD4

Provider product page for 25-0048-42

Flow cytometry

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Antibody data

Antibody Data
Antigen structure
References [25]
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Validations
Flow cytometry [1]
Other assay [24]

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Product number: 25-0048-42 - Provider product page
Provider: Invitrogen Antibodies
Product name: CD4 Monoclonal Antibody (OKT4 (OKT-4)), PE-Cyanine7, eBioscience™
Antibody type: Monoclonal
Antigen: Other
Description: Description: The OKT4 monoclonal antibody reacts with human CD4, a 59 kDa cell surface glycoprotein expressed by the majority of thymocytes, a subpopulation of mature T cells (T-helper cells) and in low levels on monocytes. CD4 is a receptor for the human immunodeficiency virus (HIV). The OKT4 antibody recognizes a different epitope than the RPA-T4 monoclonal antibody, and these antibodies do not cross-block binding to each other's respective epitopes. Applications Reported: This OKT4 (OKT-4) antibody has been reported for use in flow cytometric analysis. Applications Tested: This OKT4 (OKT-4) antibody has been pre-titrated and tested by flow cytometric analysis of normal human blood peripheral blood cells. This can be used at 5 µL (0.125 µg) per test. A test is defined as the amount (µg) of antibody that will stain a cell sample in a final volume of 100 µL. Cell number should be determined empirically but can range from 10^5 to 10^8 cells/test. Light sensitivity: This tandem dye is sensitive to photo-induced oxidation. Please protect this vial and stained samples from light. Fixation: Samples can be stored in IC Fixation Buffer (Product # 00-822-49) (100 µL of cell sample + 100 µL of IC Fixation Buffer) or 1-step Fix/Lyse Solution (Product # 00-5333-54) for up to 3 days in the dark at 4°C with minimal impact on brightness and FRET efficiency/compensation. Some generalizations regarding fluorophore performance after fixation can be made, but clone specific performance should be determined empirically. Excitation: 488-561 nm; Emission: 775 nm; Laser: Blue Laser, Green Laser, Yellow-Green Laser. Filtration: 0.2 µm post-manufacturing filtered.
Reactivity: Human
Host: Mouse
Isotype: IgG
Antibody clone number: OKT4 (OKT-4)
Vial size: 100 Tests
Concentration: 5 µL/Test
Storage: 4° C, store in dark, DO NOT FREEZE!

CD4 protein structure - 25-0048-42 shown in red.

Supportive validation

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Supportive validation

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Experimental details: NULL

Supportive validation

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Experimental details: NULL

Supportive validation

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Experimental details: NULL

Supportive validation

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Experimental details: NULL

Supportive validation

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Experimental details: NULL

Supportive validation

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Experimental details: NULL

Supportive validation

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Experimental details: NULL

Supportive validation

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Experimental details: NULL

Supportive validation

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Experimental details: NULL

Supportive validation

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Experimental details: NULL

Supportive validation

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Supportive validation

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Experimental details: Figure 7 Effects of TEX on protein expression and functions of T cells. In ( a ) down-regulation of CD69 protein expression on the surface of responder CD4 + Tconv after co-incubation with TEX. Activated CD4 + Tconv were co-incubated with TEX (10 ug protein) produced by the PCI-13 cells or with PBS for 12 h. The CD69 expression levels on CD4 + Tconv were then determined by flow cytometry (MFI) and were converted into MESF units based on calibration curves established with fluorescent calibration beads. The bar graphs show data (mean values +- SD) from 3 independent experiments performed with CD4 + Tconv obtained from different normal donors. The asterisks indicate p values at p < 0.0005. In ( b ) changes in expression levels of CD39 protein on the surface of resting CD4 + CD39 + Treg co-incubated with TEX produced by the PCI-13 cell line or DEX. The exosomes were used at the concentration of 10 ng protein/ assay. Exogenous ATP was added as described in Methods. Flow cytometry ( right ) shows up-regulation of MFI for CD39 in a representative experiment, and the bar graph summarizes results of three experiments performed with Treg obtained from different donors. In ( c ), Production levels of 5' AMP, adenosine and inosine by resting CD4 + CD39 + Treg co-incubated with TEX produced by the PCI-13 cell line. The data are from one of two experiments performed in the presence of exogenous ATP. The analyte levels were measured by mass spectrometry as described in Methods.

Supportive validation

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Experimental details: Figure 1. PS Expression on Immune Subsets in the Tumor Increases after RT C57BL/6 mice were injected with 100,000 B16F10 melanoma cells intradermally on the hindlimb. 10 days later, tumor-bearing hindlimbs received 15 Gy RT. 10 days thereafter, tumors were excised and analyzed by flow cytometry. (A) Schema and representative plots showing caspase-3/7 activity and PS expression on live (blue) versus dead (gray) tumor cells determined by a viability dye. (B) Representative gating strategy used to measure PS expression using viable CD8+ T cells from the tumor as an example. Gating was based on caspase-3/7 and annexin V fluorescence minus one (FMO). (C) Relative expression of PS on viable immune cell subsets in the tumors of mice with and without tumor-directed RT. (D) Frequencies of immune cells as a percentage of live CD45+ cells in the tumor with and without tumor-directed RT. The tables next to the figure legend lists the percentages shown in the pie charts. (E) Quantification of annexin V+ immune and tumor cells +-SEM (3-5 mice per group) from control tumors (white bars) and RT tumors (black bars). *p < 0.05.

Supportive validation

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Experimental details: Figure 2. EV quantification and cell origin in association with HIV. Purified plasma EVs fromuninfected (Control, n = 8) and HIV+ patients (n = 17) were stained with the lipophilic fluorescent tracer dye DiD to count total vesicles ( Figure 2A and B ), then labeled with antibodies directed against receptors CD45 ( Figure 2C and D ), CD4 ( Figure 2E and F ), and CD8 ( Figure 2 G and H ) to evaluate cell origin by cytofluorometry. Asterisks denote significant difference (** P

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Experimental details: Figure 7 HEBCan Rescues Hematopoiesis and T Cell Development in HEB -/- hESCs in OP9-DL4 Co-cultures (A) qRT-PCR analysis for the expression of hematopoietic genes in CD34 + cells sorted from WT, KO + GFP, and KO + HEBCan day-8 (d8) EBs. mRNA levels are shown relative to GAPDH. (B and C) Percentages (B) and numbers (C) of CD45 + cells in d12 and d18 OP9-DL4 co-cultures. (D and E) Flow-cytometric analysis of T cell development from WT, KO + GFP, and KO + HEBCan d8 EB-derived CD34 + cells at d12 (D) and d18 (E) of OP9-DL4 co-culture. Cells are gated on the CD45 + DAPI - population. Error bars represent mean +- SD (n = 3 independent experiments). * p < 0.05, ** p < 0.01, *** p < 0.005 by Student's t test. Plots in (B), (D), and (E) are representative of three independent experiments.

Supportive validation

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Experimental details: Figure 1. Flow cytometric analysis was used to determine the distribution of Th22, Th17 and Th1 cells in EGC, HE and HY ( Fig. 1 ). Flow cytometric analysis of Th22, Th17 and Th1 cells in peripheral whole blood from EGC (n=39), HE (n=32) and HY (n=31). (A) Lymphocytes were gated in P1 using flow cytometry. CD4 + IFN-gamma - lymphocytes were gated in P2 using flow cytometry, and representative results of flow cytometric analyses for (B) Th1 (CD4 + IFN-gamma + ), (C) Th22 (CD4 + IFN-gamma - IL-17 - IL-22 + ) and Th17 (CD4 + IFN-gamma - IL-17 + IL-22 - ) cells in the three groups of subjects are presented. The number of cells stained in EGC, HE and HY in P2 were 2,654, 4,696 and 5,185, respectively. The proportion of (D) Th22, (E) Th17 and (F) Th1 cells in the three groups of subjects. The proportion of (G) Th22 and (H) Th17 cells in peripheral whole blood derived from patients with early (n=13) or advanced (n=26) gastric cancer. The association between the proportion of (I) Th22 and Th17 cells, (J) Th22 and Th1 cells, and (K) Th17 and Th1 cells, in peripheral whole blood of all subjects. *P

Supportive validation

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Experimental details: Fig. 1 CD46 co-stimulation provides superior support for CTL activity. a CD46 expression on the surface of resting human CD4 + and CD8 + T cells assessed by FACS analysis ( n = 4, gating strategy in Supplementary Fig. 7a ). b Comparison of IFN-gamma, TNF-alpha and IL-10 secretion by CD3 + CD46-activated T cells. Purified CD4 + and CD8 + T cells from healthy donors were left non-activated (NA) or stimulated with immobilized antibodies to CD3, CD3 + CD28 or CD3 + CD46 and cytokines measured 60 h post activation ( n = 5). c, d Degranulation (CD107a staining, ( c )) and granzyme B expression ( d ) by CD8 + T cells upon CD46 co-stimulation. CD8 + T cells were stimulated as in ( a ) and CD107a and granzyme B expression assessed with left panels showing representative cytometry images and right panels corresponding quantifications ( n = 3, gating strategy in Supplementary Fig. 7b ). e Killing activity of CD46-activated CD8 + T cells. T cells were stimulated as depicted for 24 h and cytotoxic activity of differently activated CD8 + T cells towards DU145 target cells assessed 24 h post co-culture of T cells and DU145 cells ( n = 4, gating strategy in Supplementary Fig. 7c ). f Effect of CD46 co-stimulation on CD8 + T-cell proliferation. Purified T cells were activated as indicated for 5 days and cell proliferation measured each day ( n = 4) (black circles, non-activated cells; green, blue, and red circles, CD3, CD3 + CD28 or CD3 + CD46-activated cells, res

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Experimental details: Fig. 1 CD20 dim CD4 + T cells involve memory phenotypes with more activation than CD20 - cells. PBMC from uninfected donors, ART-suppressed patients and viremic patients were stained with CD20, activation markers (HLA-DR, CD69, CD25), and T-cell differentiation markers (CCR7, CD45RO, CD27, CD95). a Gating strategy used to identify CD20 dim CD4 + T cells. Previous sequential gates are represented in Supplementary Fig. 9 . Viable CD4 + T cells (identified by CD3 and CD4 expression) were first selected for the analysis of CD20 expression, defined by its Fluorescence Minus One (FMO) control. T-cell memory subsets were selected as follows: CD4 + T NA (CCR7 + , CD45RO - , CD27 + , CD95 - ), CD4 + T SCM (CCR7 + , CD45RO - , CD27 + , CD95 + ); CD4 + T CM (CCR7 + , CD45RO + ), CD4 + T TM (CCR7 - , CD45RO + , CD27 + ); CD4 + T EM (CCR7 - , CD45RO + , CD27 - ), CD4 + T TD (CCR7 - , CD45RO - ). b , c Representative bright-field and pseudo-color fluorescence images of CD20 dim CD4 + T cells ( b ) and B cells ( c ) from two ART-suppressed patients (#9 and #22) using the Amnis imaging flow cytometer technology. Scale bar 10 um. d Percentage of CD20 dim expression within CD4 + T cells in uninfected controls and the two patient cohorts. Mann-Whitney comparison was used to compare n = 6 uninfected controls, n = 21 ART-suppressed patients, n = 20 viremic patients. e Expression of the activation markers CD25, CD69 or HLA-DR in CD20 dim and CD20 - CD4 + T cells in different cohorts of HIV + patie

Supportive validation

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Experimental details: Fig. 5 Ex vivo infection upregulates CD20 expression. Unstimulated PBMCs from three uninfected donors were infected with HIV strain NL4.3. Infection was monitored by simultaneous staining of HIV RNA using the RNA FISH-flow assay and the viral protein p24 at days 3, 4, and 5. a Gating strategy used to monitor HIV infection and expression of CD20 dim . Previous sequential gates are represented in Supplementary Fig. 9 . b Percentage of productively HIV-infected cells in CD3 + T cells (left panel) and in CD20 dim or CD20 - CD3 + T cells (right panel). c Expression of CD20 dim in infected and uninfected CD3 + T cells (left panel) and in the total CD3 + T cell population (right panel). d Correlation between the proportion of infected cells within the CD3 + T cell population and CD20 expression (left panel) and the mean fluorescence intensity (MFI) of CD20 (right panel). e Percentage of HLA-DR and PD-1 in infected and uninfected cells expressing or not expressing CD20 dim . f Proportion of CD20 dim in infected cells expressing the CD4 cell receptor versus infected cells with marked downregulation of the CD4 receptor. In all panels, the mean and SEM value of three independent experiments is represented. In panel ( d ), Spearman's nonparametric correlation coefficients and associated p values are shown. Data underlying this Figure are provided as Source Data file

Supportive validation

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Experimental details: Figure 1 Anti-HER2 DARPin-Targeted CAR-T Cells Demonstrated Similarl Efficacy In Vitro (A) Schematics of the dual-promoter lentiviral (LV) gene cassettes used to generate anti-HER2 DARPin-targeted first- or second-generation CAR-T cells (structural details are as indicated; TM, transmembrane; IC, intracellular) or CAR-negative control NGFR-T cells. In all cases, truncated NGFR (tNGFR) is included as a transduction marker. (B) Expression of CARs on the surface of engineered (NGFR + ) T cells as determined by flow cytometry (upstream gating strategy: lymphocytes - singlets - NGFR + ). Mean fluorescence intensity (MFI) for CAR expression is indicated in brackets. Representative results have been replicated in 2-4 additional independent experiments. (C and D) Production of IFN-gamma (C) and TNF-alpha (D) upon CAR-T cell stimulation with HER2 + (OVCAR-3; closed symbols) or HER2 - (LOX-IMVI; open symbols) human tumor cell lines was measured by intracellular cytokine staining (ICS) and subsequent flow cytometry (upstream gating strategy: lymphocytes - singlets - CD4 + or CD8 + T cells). Percent cytokine production was normalized for transduction (transduction ranges observed: DARPin-28z, 39%-60%; DARPin-BBz, 33%-52%; DARPin-z, 25%-63%; NGFR, 63%-86%). Each point indicates data from a single independent experiment (n = 3-5 per LV construct); black lines indicate mean values. (E and F) Cytotoxicity across various effector:target (E:T) ratios with LOX-IMVI (E) or OVCAR-3 (F) tumor cell

Supportive validation

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Experimental details: Figure 3 DAPRin-28z-T Cells Activated in the Lungs and Heart, Resulting in a Systemic Cytokine Storm OVCAR-3 tumor-bearing NRG mice were treated with 6 x 10 6 effective DARPin-28z-T cells or a matched number of NGFR-T cells. (A-C) Mice were sacrificed at 1, 3, or 5 days post-ACT1 for total body perfusion, fixation, necropsy, and histological analysis. (A) Hematoxylin and eosin (H&E) staining of the lungs at 20x magnification (scale bars, 100 mum). V, vasculature. (B) Immunohistochemistry (IHC) for human CD3 in the lungs at 20x magnification (scale bars, 100 mum) or 60x magnification (zoom-in; scale bars, 50 mum). (C) H&E or CD3 IHC staining of the heart at 20x magnification (scale bars, 100 mum); arrow indicates aberrant region of inflammation along the right heart wall. Representative images from n = 2-3 mice are shown. Findings have been recapitulated in 1-2 additional independent experiments. (D) DARPin-28z- or NGFR-T cells were co-cultured with tumor-free NRG mouse lung homogenates ex vivo . T cell proliferation was measured by flow cytometry using CellTrace Violet (CTV) dye. Data are representative of two independent experiments. (E) Mice were bled at 1, 3, 5, or 7 days post-ACT1 for multiplex analysis of human serum cytokine content; a globally normalized heatmap of log2-transformed human cytokine fluorescence readings is shown. Each square represents data from one mouse. Colorimetric scale bar indicates minimum, average, and maximum values on map. Absolute values are d

Supportive validation

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Experimental details: Figure 4 Differential In Vivo Toxicity of DARPin-28z-T Cells Manufactured from Unique PBMC Donors Correlated with the Frequency of CD4+ T Cells in the Adoptive Transfer Product (A) OVCAR-3 tumor-bearing NRG mice were treated with 6.0 x 10 6 or 1.7-2.0 x 10 6 DARPin-28z-T cells produced from MAC026, LEUK001, or MAC014 PBMCs. Mice were monitored over time for changes in weight. Data were pooled from n = x independent experiments. For 6.0 x 10 6 cells, MAC014, 2; LEUK001, 3; and MAC026, 4. For 2.0 x 10 6 cells, MAC014, 1; LEUK001, 2; and MAC026, 1. Each line indicates data from one animal; curves end, indicating when mice succumbed to toxicity. (B) Composition of CD4 + or CD8 + cells in DARPin-28z-T cell products (days 13-14 post-activation) manufactured using starting PBMCs from donors as indicated and determined using flow cytometry (upstream gating strategy: lymphocytes - singlets - NGFR + ). Error bars represent SD. Data from n = x independent experiments; MAC014, 5 (2 unique PBMC preparations); LEUK001, 6 (1 PBMC preparation); and MAC026, 12 (5 unique PBMC preparations).

Supportive validation

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Experimental details: Figure 6 Enhanced proliferation and IFNgamma release of CD4+ T cells induced by moDCs stimulated with supernatant of NB-PDT treated tumor cells. A431 cells were treated with NB-PDT, the supernatant was collected 24 h later and incubated with immature moDCs for another 24 h. moDCs were then co- incubated with allogeneic CFSE-labeled CD4+ T cells in a 1:10 ratio. After 6 days, CD4+ T cell proliferation was measured with flow cytometry and IFNgamma release was assessed by ELISA. ( a ), Percentage of CD4+ T cells with weak CFSE signal, thus proliferating cells (n = 4). ( b ), Quantification of released IFNgamma by CD4+ T cells (n = 3). Each combination of allogeneic donors is represented by a different symbol and color. ctr, unstimulated DCs; LPS, LPS-stimulated DCs; NT, untreated tumor cells; LD50, mild cytotoxic NB-PDT; LD100, highly cytotoxic NB-PDT; Light, only light control; NB-PS, only NB-PS conjugate control. Significance is displayed as * p