α cd4

Manufactured by BioXCell

Sourced in Japan

α-CD4 is a laboratory equipment used to detect and quantify the presence of CD4+ T cells in biological samples. It functions by specifically binding to the CD4 receptor on the surface of T cells, allowing for their identification and enumeration.

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7 protocols using α cd4

T cell depletion in mice

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Depletion antibodies were obtained from BioXCell: α-mouse CD4 (GK1.5, BP0003-1), α-mouse CD8a (2.43, BP0061), and rat IgG2b isotype control (LTF-2, BP0090). Mice were given intraperitoneal injections one day prior to infection and at days 7, 14, and 21 post infection. For depletions of both CD4⁺ and CD8⁺ T cell subsets, mice were treated with 500 μg α-CD4 and 500 μg α-CD8, or 1 mg isotype control, in a 500 μl volume of pH 7.0 dilution buffer (BioXCell). For single subset depletions, mice received 500 μg α-CD4 or 500 μg α-CD8, or 500 μg isotype control, in a 250 μl volume of pH 7.0 dilution buffer (BioXCell). T cell depletions were performed only during the initial infection, not the challenge infection.

O’Brien V.P., Dorsey D.A., Hannan T.J, & Hultgren S.J. (2018). Host restriction of Escherichia coli recurrent urinary tract infection occurs in a bacterial strain-specific manner. PLoS Pathogens, 14(12), e1007457.

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LCMV Clone 13 Infection in Mice

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Mice were infected with 4 × 10⁶ PFU LCMV Clone 13 i.v., monitored for weight loss, and bled or sacrificed at day 8, 15, or 30 post infection for flow cytometry analyses. For viral titer studies, mice were bled at days 8, 15, and 30 post infection. Liver lymphocytes were isolated by dissociation of the liver followed by a 40%/60% Percoll gradient. Lung lymphocytes were isolated by dissociation of the lung on a gentleMACS Dissociator followed by a 37 °C incubation in collagenase for 30 min. Lymphocytes were enriched on a 40%/60% Percoll gradient. To deplete CD4⁺ T cells, mice were injected i.p. with 200 µg αCD4 (BioXCell Cat# BE0003-1) on days -1 and 1 (relative to LCMV Clone 13 injection on day 0).

LaFleur M.W., Nguyen T.H., Coxe M.A., Yates K.B., Trombley J.D., Weiss S.A., Brown F.D., Gillis J.E., Coxe D.J., Doench J.G., Haining W.N, & Sharpe A.H. (2019). A CRISPR-Cas9 delivery system for in vivo screening of genes in the immune system. Nature Communications, 10, 1668.

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Evaluating Immunotherapies in Influenza Infection

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Influenza-infected WT mice were administrated with control IgG or various neutralizing or depleting Ab as described in the related Results sections. For CD4⁺ T cell depletion with high dose of α-CD4, mice were injected with 250 μg of α-CD4 weekly (clone: GK1.5, BioXCell) starting at 14 d.p.i. For circulating CD4⁺ T cell depletion, mice were intraperitoneally injected with 40 μg of α-CD4 for the first dose followed with 10 μg of α-CD4 weekly. CD40L blockade was achieved by the injection of 250 μg of α-CD40L (clone: MR-1, BioXCell) weekly, respectively. For systemic IL-21R blockade, 500 μg of α-IL21R (clone: 4A9, BioXCell) was injected intraperitoneally weekly starting at 14 d.p.i. For lung local IL-21R blockade, 50 μg of α-IL21R was injected through intranasal route weekly starting at 14 d.p.i. In some experiments, FTY720 (1 mg/kg; Cayman) was administrated via intraperitoneal injection daily from 13 d.p.i. to block lymphocyte migration until mouse euthanasia.

Son Y.M., Cheon I.S., Wu Y., Li C., Wang Z., Gao X., Chen Y., Takahashi Y., Fu Y.X., Dent A.L., Kaplan M.H., Taylor J.J., Cui W, & Sun J. (2021). Tissue-resident CD4+ T helper cells assist the development of protective respiratory B and CD8+ T cell memory responses. Science immunology, 6(55), eabb6852.

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Studying Malaria Immune Modulation in Mice

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C57BL/6 mice (8 weeks, 18–22 g) were purchased from Jackson Laboratories and housed in the Biomedical Sciences Building at OUHSC. The OUHSC IACUC approved all experiments. Plasmodium yoelii (clone 17XNL, obtained from MR4, ATCC) was routinely passaged through mosquitoes and mouse infections were initiated by serial transfer of 10⁶ parasite-infected red blood cells via tail vein injection. Parasitemia was measured using flow cytometry as described (Malleret et al., 2011 (link)). Giemsa staining of thin blood smears was done in parallel. At the indicated times, mice were injected i.p. with 200 μg α-CD4 (GK1.5), 500 μg of α-IFN-γ (XMG1.2), 200 μg α-PD-L1 (10F.9G2), 50 μg of α-OX40 Ab (OX86), 200 μg α-PD-L1 and 50 μg α-OX40, or 200 μg α-PD-1 (RMP1-14) and 50 μg α-OX40, or equivalent amounts of rat IgG. All biologics were acquired from BioXcell. Recombinant IFN-γ was acquired from Tonbo.

Zander R.A., Obeng-Adjei N., Guthmiller J.J., Kulu D.I., Li J., Ongoiba A., Traore B., Crompton P.D, & Butler N.S. (2015). PD-1 co-inhibitory and OX40 co-stimulatory crosstalk regulates helper T cell differentiation and anti-Plasmodium humoral immunity. Cell host & microbe, 17(5), 628-641.

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Lm-OVA Immunization and Depletion

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Mice were immunized with different strategies using OVA-expressing Lm strains. 30 days following primary immunization, mice were re-challenged by oral gavage with 2 × 10¹⁰ LmOVA. Mice were euthanized 3 days post infection and harvested organs were homogenized and plated.
For depletion experiments, mice were treated on day −3, −1 and +1 (with 0 being the day of re-challenge) with the following antibodies: α-CD8α (Clone 2.43, 500 μg), α-CD4 (GK1.5, 200 μg) α-γδ TCR (UC7, 100 μg) (all from BioXCell) similarly to what previously published^{36 (link)}. Mice were sacrificed 3 days post re-challenge and organs plated.

Becattini S., Littmann E.R., Seok R., Amoretti L., Fontana E., Wright R., Gjonbalaj M., Leiner I.M., Plitas G., Hohl T.M, & Pamer E.G. (2020). Enhancing mucosal immunity by transient microbiota depletion. Nature Communications, 11, 4475.

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Combination Immunotherapy and Chemotherapy for Cancer

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Antibodies were delivered intraperitoneally in 100 μl of phosphate-buffered saline (PBS). αCD4 (catalog no. GK1.5, BioXCell) and αCD8 (catalog no. 2.43, BioXCell) were administered at 200 μg every 4 d. αPD-1 (catalog no. 29F.1A12, BioXCell) was administered at 200 μg 3× a week. αCTLA (catalog no. 9H10, BioXCell) was administered at an initial dose of 200 μg, with subsequent doses at 100 μg, 3× a week. Oxaliplatin (Sigma-Aldrich) and cyclophosphamide (Sigma-Aldrich) (Oxa/Cyc) were co-delivered intraperitoneally in 100 μl of PBS at 2.5 mg per kg body weight and 50 mg per kg body weight, respectively, once a week for 3 weeks, as previously described^{42 (link)}.

Westcott P.M., Muyas F., Hauck H., Smith O.C., Sacks N.J., Ely Z.A., Jaeger A.M., Rideout WM I.I.I., Zhang D., Bhutkar A., Beytagh M.C., Canner D.A., Jaramillo G.C., Bronson R.T., Naranjo S., Jin A., Patten J.J., Cruz A.M., Shanahan S.L., Cortes-Ciriano I, & Jacks T. (2023). Mismatch repair deficiency is not sufficient to elicit tumor immunogenicity. Nature Genetics, 55(10), 1686-1695.

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Immune Cell Depletion Experiments

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For investigation of the role of different immune cell types, functional depletion experiments were carried out. Mice were given Panc02 or KPC tumors as described above. CD4⁺ and CD8⁺ T cells were depleted using 200 µg of αCD4 (Clone GK1.5, BioXCell) or αCD8 (Clone YTS 196.4, BioXCell) monoclonal antibodies, respectively, and NK cells were depleted using 25 µL of anti‐asialo‐GM1 (WAKO, Osaka, Japan) per mice. Mice were randomised into six different treatment groups: (1) Isotype control; (2) IL‐15 + αCD40; (3) IL‐15 + αCD40 + αCD4; (4) IL‐15 + αCD40 + αCD8; (5) IL‐15 + αCD40 + αNK; and (6) IL‐15 + αCD40 + αCD8 + αNK. Depletion antibodies or anti‐asioalo‐GM1 were given i.p. at days −1, 0, 3, 6, 10 and 14. Tumor kinetics and survival were measured as described above.

Van Audenaerde J.R., Marcq E., von Scheidt B., Davey A.S., Oliver A.J., De Waele J., Quatannens D., Van Loenhout J., Pauwels P., Roeyen G., Lardon F., Slaney C.Y., Peeters M., Kershaw M.H., Darcy P.K, & Smits E.L. (2020). Novel combination immunotherapy for pancreatic cancer: potent anti‐tumor effects with CD40 agonist and interleukin‐15 treatment. Clinical & Translational Immunology, 9(8), e1165.

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