47-0257-42

antibody from Invitrogen Antibodies

Targeting: IL2RA CD25, IDDM10, IL2R

Flow cytometry (Supportive data in Antibodypedia)

Other assay (Supportive data in Antibodypedia)

Antibody Data

Product number

47-0257-42

Provider

Invitrogen Antibodies

Product name

CD25 Monoclonal Antibody (CD25-4E3), APC-eFluor™ 780, eBioscience™

Antibody type

Monoclonal

Antigen

Other

Description

Description: This CD25-4E3 monoclonal antibody reacts to human CD25, which is also known as the low-affinity interleukin (IL)-2 receptor alpha. CD25 is expressed on activated T cells (including a subset of regulatory T cells), B cells, and macrophages. This receptor subunit associates with CD122, the IL-2 receptor beta chain, and CD132, the common gamma chain, to form the high-affinity IL-2 receptor. IL-2 receptor signaling has been linked to cell proliferation and survival in lymphocytes. CD25 is a marker for Hodgkin's disease, and has been associated with such autoimmune diseases as Type I diabetes. This monoclonal antibody is cross-reactive to rhesus monkey. Applications Reported: This CD25-4E3 antibody has been reported for use in flow cytometric analysis. Applications Tested: This CD25-4E3 antibody has been pre-titrated and tested by flow cytometric analysis of normal human peripheral blood cells. This can be used at 5 µL (0.03 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. APC-eFluor® 780 emits at 780 nm and is excited with the Red laser (633 nm). Please make sure that your instrument is capable of detecting this fluorochome. 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 # 00-8222) (100 µL of cell sample + 100 µL of IC Fixation Buffer) or 1-step Fix/Lyse Solution (Product # 00-5333) 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: 633-647 nm; Emission: 780 nm; Laser: Red Laser. Filtration: 0.2 µm post-manufacturing filtered.

Reactivity

Human

Host

Mouse

Isotype

IgG

Antibody clone number

CD25-4E3

Vial size

100 Tests

Concentration

5 µL/Test

Storage

4° C, store in dark, DO NOT FREEZE!

References

Submitted references

Increased TNF-α Initiates Cytoplasmic Vacuolization in Whole Blood Coculture with Dengue Virus.

Satria RD, Huang TW, Jhan MK, Shen TJ, Tseng PC, Wang YT, Yang ZY, Hsing CH, Lin CF
Journal of immunology research 2021;2021:6654617

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Remote Limb Ischemic Postconditioning Protects Against Ischemic Stroke by Promoting Regulatory T Cells Thriving.

Yu HH, Ma XT, Ma X, Chen M, Chu YH, Wu LJ, Wang W, Qin C, Tian DS
Journal of the American Heart Association 2021 Nov 16;10(22):e023077

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Murlentamab, a Low Fucosylated Anti-Müllerian Hormone Type II Receptor (AMHRII) Antibody, Exhibits Anti-Tumor Activity through Tumor-Associated Macrophage Reprogrammation and T Cell Activation.

Prat M, Salon M, Allain T, Dubreuil O, Noël G, Preisser L, Jean B, Cassard L, Lemée F, Tabah-Fish I, Pipy B, Jeannin P, Prost JF, Barret JM, Coste A
Cancers 2021 Apr 13;13(8)

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HDAC Inhibitor, CG-745, Enhances the Anti-Cancer Effect of Anti-PD-1 Immune Checkpoint Inhibitor by Modulation of the Immune Microenvironment.

Kim YD, Park SM, Ha HC, Lee AR, Won H, Cha H, Cho S, Cho JM
Journal of Cancer 2020;11(14):4059-4072

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ATP Release from Chemotherapy-Treated Dying Leukemia Cells Elicits an Immune Suppressive Effect by Increasing Regulatory T Cells and Tolerogenic Dendritic Cells.

Lecciso M, Ocadlikova D, Sangaletti S, Trabanelli S, De Marchi E, Orioli E, Pegoraro A, Portararo P, Jandus C, Bontadini A, Redavid A, Salvestrini V, Romero P, Colombo MP, Di Virgilio F, Cavo M, Adinolfi E, Curti A
Frontiers in immunology 2017;8:1918

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Tumor specific regulatory T cells in the bone marrow of breast cancer patients selectively upregulate the emigration receptor S1P1.

Rathinasamy A, Domschke C, Ge Y, Böhm HH, Dettling S, Jansen D, Lasitschka F, Umansky L, Gräler MH, Hartmann J, Herold-Mende C, Schuetz F, Beckhove P
Cancer immunology, immunotherapy : CII 2017 May;66(5):593-603

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HDAC inhibition potentiates immunotherapy in triple negative breast cancer.

Terranova-Barberio M, Thomas S, Ali N, Pawlowska N, Park J, Krings G, Rosenblum MD, Budillon A, Munster PN
Oncotarget 2017 Dec 26;8(69):114156-114172

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HLA Class II tetramers reveal tissue-specific regulatory T cells that suppress T-cell responses in breast carcinoma patients.

Schmidt HH, Ge Y, Hartmann FJ, Conrad H, Klug F, Nittel S, Bernhard H, Domschke C, Schuetz F, Sohn C, Beckhove P
Oncoimmunology 2013 Jun 1;2(6):e24962

Flow cytometry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Staining of normal human peripheral blood cells with Anti-Human CD4 PE (Product # 12-0049-42) and Mouse IgG2b K Isotype Control APC-eFluor® 780 (Product # 47-4732-80) (left) or Anti-Human/Non-Human Primate CD25 APC-eFluor® 780 (right). Cells in the lymphocyte gate were used for analysis.

Other assay

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

NULL

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Fig. 1 Decreased frequencies of Treg subpopulations in the BM of breast cancer patients. a Representative plot of a healthy donor showing the CD4+ T-cell gating strategy. b-c CD4+ cells were analyzed for FoxP3 and CD25 expression. CD25+ FoxP3+ cells were gated as Treg, and CD25- FoxP3- cells were gated as Tcon. Representative plots illustrating Treg frequencies in BM and PB of a healthy donor ( b ) and a breast cancer patient ( c ). Red circle and square represents the CD4 gate and CD25+ FoxP3+ Treg gate, respectively. d Cumulative data of CD25+ FoxP3+ Treg frequencies in PB and BM of all patients and healthy donors analyzed--Healthy donor PB ( n = 7) and BM ( n = 8), patients with matched PB and BM samples ( n = 50). Data distribution in 1 d is represented by mean with SEM. For healthy donors unpaired t test and for patient samples, paired t test was used for statistical analysis. Epigenetic PCR was performed on DNA isolated from tumor areas from FFPE sections obtained from 42 patients. Samples that passed quality control [Treg ( n = 33) and CD3 ( n = 40)] were taken into analysis. e, f Treg and CD3 T-cell percentage ( e ) and counts per mm 3 volume of tumor ( f ). Data distribution is represented by median with interquartile range in e and f. g Graph bridging data of Treg frequencies in BM, PB, and tumor--ratio of Treg in BM to PB correlated to ratio of Treg in tumor to PB [for all patients with Treg frequencies >4.6% in PB ( n = 18)]. Non-parametric Spearman Correlation wa

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Figure 4 Murlentamab opsonization of SKOV3-R2 + activates an effective anti-tumor T cell immune response. SKOV3-R2 + ovarian tumor cells were labeled with different 3C23K antibodies (3C23K-FcKO control, 3C23K-CHO normally fucosylated or murlentamab the low fucosylated form) and cultured in the presence of human monocyte-derived macrophages from healthy donors unstimulated (M0) or stimulated with M-CSF and IL-10 (TAMs). After 3 days of co-culture, activated T cells coming from the same healthy donor were added in the culture well for 4 more days. ( A ) The CD4 + Th1/Th2 polarization profile, ( B ) the proportion of CD3 + CD4 + CD25 + regulatory T cells and ( C ) the activation of T CD8 + cells were determined by flow cytometry after four days of co-culture. Data shown (boxplots) are the results from two different experiments (performed with two different healthy donors). * p < 0.05; ** p < 0.01; *** p < 0.001. p values were determined using one-way ANOVA analysis followed by Tukey''s multiple comparisons test.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Figure 3. Tetramer staining of conventional and regulatory T cells from breast cancer patients. ( A-G ) Peripheral blood mononuclear cells (PBMCs) of a breast cancer patient were analyzed, upon gating on living CD3 + CD4 + T cells. The patient sample was stained with tetramers presenting either mam 34-48 ( B ) or the CLIP peptide ( C ). Numbers indicate the percentage of cells in the respective gate, referring to lymphocytes ( A ) or CD3 + CD4 + T cells ( B ) and ( C ). Within CD3 + CD4 + T cells, regulatory T cells (Tregs) were identified as CD25 high CD127 low ( D ) and ( G ) and CD25 high CD127 low FOXP3 + ( E ), ( F ) and ( H ). ( G ) reports the percentage of Tregs within tetramer-positive cells in a representative patient. ( I ) Frequency of mam 34-48 - and CLIP-presenting tetramer-positive cells among CD3 + CD4 + T cells of breast cancer patients and healthy donors (HD) (p values as per Mann-Whitney U tests are indicated). ( J and K ) Frequency of mam 34-48 -specific Tcon ( J ) cells and Tregs ( K ) of breast cancer patients and HDs.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Figure 4 Immune profiling in DENV-treated whole blood cells 24 h postincubation. Following DENV (MOI = 1) coculture in 100 mu l of WB ex vivo for 24 h, (a) representative flow cytometric analysis and gating of various cells obtained from five cases, performed by staining for specific cell surface markers (CD4, CD8, CD11c, CD14, CD16, CD19, CD25, CD56, CD62L, and HLA-DR), in the DENV-infected and mock groups showed (b) the changes in the expression of specific immune cell populations as noted. (c) The results are shown as a percentage of the mean +- SD obtained from five cases. * p < 0.05, ** p < 0.01, and *** p < 0.001, compared to the mock group. R: region; WBC: white blood cell; bri: bright.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Figure 2 RLIPoC increases Tregs abundance in the blood, spleen, and brain. A , Representative flow cytometry plots of CD4 + CD25 + Foxp3 + Tregs in the blood and spleen on day 1, 3, and 7 after MCAO. B , Quantification of percentages of CD25 + Foxp3 + Tregs among CD4 + T cells in the blood and spleen. Student t test, * P

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Figure 3 RLIPoC facilitated Tregs increase via modifying metabolic spectrum. A , Images of blood flow (top) and curves of the dynamic concentration changes of metabolites (FAD, Cytaa3-R, and Cytc-R) in the region of the hind limb underwent RLIPoC in vivo, which was monitored using a multispectral optical imaging system. O indicates occlusion of the bilateral femoral arteries; and R, reperfusion of the bilateral femoral arteries. B , Concentrations of NAD + and NADH, and the ratio of NAD + to NADH, in muscle that underwent RLIPoC. Student t test, * P

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Figure 4 Depletion of Tregs in vivo abolished neuroprotection conferred by RLIPoC. A , Scheme for ablation of Tregs in DEREG mice (left) and purified Tregs transferring to recipient mice (right). Diphtheria toxin (DT; intraperitoneal, 50 ug/kg body weight) was administered 2 days before the MCAO model and was administered again with a dose of 10 ug/kg body weight every other day until the mouse was euthanized. Purified CD4 + CD25 + Tregs were transferred intravenously to recipient mice within 1 hour after the MCAO model, with a dose of 1 million per mouse through the tail vein. B , The efficacy of Tregs ablation in DEREG mice using DT. Representative flow cytometry plots and the quantitation of percentages of CD25 + Foxp3 + Tregs among CD4 + T cells in the blood and spleen are shown in the histogram. Student t test, * P

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Figure 3 CG-745 increases helper T cells, cytotoxic T cells and natural killer T cells, and decreases Treg: (A) hPBMCs were incubated with CG (CG-745) for 36 hours and a subset of hPBMCs was analyzed using the antibodies indicated in the text; (B) hPBMCs were co-cultured with Huh7, Hep3B, HepG2 or PLC/PRF/5 cells for 36 hours with or without CG, and a subset of hPBMCs was analyzed by Attune Nxt (Invitrogen, USA).