Anti nk1.1 clone pk136

Manufactured by BioXCell

Sourced in United States

Anti-NK1.1 (clone PK136) is a mouse monoclonal antibody that recognizes the NK1.1 antigen expressed on natural killer (NK) cells and a subset of T cells in mice. It can be used for the identification and characterization of NK1.1-positive cell populations.

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37 protocols using anti nk1.1 clone pk136

NK1.1 and CD1d Modulation in 3xTg-AD Mice

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3xTg-AD and control B6129SF2/J were obtained from MMRRC JAX or the Jackson Laboratory, and bred in the animal facility of Albany Medical College. 7–8 months old female mice were used in this study. For anti-NK1.1 treatment, mice were treated with 25μg anti-NK1.1 (clone PK136, Bio X Cell) antibodies or isotype control (clone C1.18.4, Bio X Cell) every 4 days for 4 weeks. For anti-CD1d treatment, mice were treated with 500μg anti-CD1d (clone 19G11, Bio X Cell) antibodies or isotype control every other day for 4 weeks. Water Maze tests were performed on the day after the last treatment. Specifically, for anti-Nk1.1 treatment, mice were treated with anti-NK1.1 antibodies or isotype controls on day 1, 5, 9, 13, 17, 21, 25, 29; and Water Maze test was performed on day 30. For anti-CD1d treatment, mice were treated with anti-CD1d antibodies or isotype control on day 1, 3, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29; and Water Maze test was performed on day 30. All animal experiments were performed according to protocols approved by the Institutional Animal Care and Use Committee at Albany Medical College.

Zhang Y., Fung I.T., Sankar P., Chen X., Robison L.S., Ye L., D’Souza S.S., Salinero A.E., Kuentzel M.L., Chittur S.V., Zhang W., Zuloaga K.L, & Yang Q. (2020). Depletion of NK cells improves cognitive function in the Alzheimer’s Disease mouse model. Journal of immunology (Baltimore, Md. : 1950), 205(2), 502-510.

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Immune Cell Depletion in Tumor Models

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Mice were administered depleting antibodies or appropriate isotype control antibodies intraperitoneally (IP) for each experiment as follows: CD8 Depletion: 250 μg of anti-CD8a clone 2.43 (BioXCell, Lebanon, NH), starting day 6 and weekly thereafter. CD4 Depletion: 250 μg of anti-CD4 clone GK1.5 (BioXCell), starting day 6 then weekly thereafter. BALB/c NK cell depletion: 20 μL of anti-asialo GM1 rabbit serum (Wako Chemicals, Richmond, VA) or control rabbit serum, starting day 6, repeated every 4 days for a total of 4 injections. BL/6 NK cell depletion: 200 μg of anti-NK1.1 clone PK-136 (BioXCell) starting day 6, repeated every 4 days for a total of 4 injections. IFN-γ depletion: 100 μg of anti-IFN- γ clone R4–6A2 (BioXCell), on days 7, 9, 15 and 21. After repeated antibody injections, some mice developed a fatal anaphylactic reaction, which correlated with tumor burden. These mice were censored from survival curves since they did not meet the experimental endpoint, and antibody treatments were discontinued for the remaining mice. When more than half of the mice in a treatment group died or were sacrificed, the group was censored from the tumor regression curves to avoid skewing the mean.

Mastria E.M., Cai L.Y., Kan M.J., Li X., Schaal J.L., Fiering S., Gunn M.D., Dewhirst M.W., Nair S.K, & Chilkoti A. (2017). Nanoparticle formulation improves doxorubicin efficacy by enhancing host antitumor immunity. Journal of controlled release : official journal of the Controlled Release Society, 269, 364-373.

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Radiation-Induced Immune Modulation

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For PD-L1 blockade, anti-PD-L1 Ab (clone 10 F.9G2, BioXcell), or rat IgG2b (clone LTF-2, BioXcell) were given intraperitoneally (i.p.) every third day from the day RT performed at a dose of 200 μg/mouse. For in vivo depletion of lymphocytes, 200 μg of anti-CD4 (clone GK1.5, BioXcell), anti-CD8β (clone Lyt 3.2, BioXcell), anti-NK1.1 (clone PK136, BioXcell) Abs, or rat IgG2b (clone LTF-2, BioXcell) Ab were injected i.p. every third day for three times from the day when RT was given. For in vivo depletion of IL-12 and IFN-γ, 1 mg of anti-IL-12p40 (clone C17.8, BioXcell) and anti-IFN-γ (clone R4⁻6A2, BioXcell) Abs were administrated by i.p. injection at the day when RT was performed, with follow-up doses of 500 μg for five consecutive days.

Oba T., Long M.D., Keler T., Marsh H.C., Minderman H., Abrams S.I., Liu S, & Ito F. (2020). Overcoming primary and acquired resistance to anti-PD-L1 therapy by induction and activation of tumor-residing cDC1s. Nature Communications, 11, 5415.

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Modulating Lymphoma with Type I IFN

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20,000U of Type I IFNα (R&D systems) was administered intraperitoneally to SRα-tTA/tet-O-MYC mice bearing overt lymphoma for 3 days. On day 3, spleens from treated mice were collected, and subjected to flow cytometry for NK and T cell analysis. PBS-treated mice were used as controls. Absolute cell counts were obtained as described in section ‘Calculation of absolute immune cell counts’. For long-term administration of Type I IFNα, the mice were treated every third day with 20,000 U IFNα or PBS intraperitoneally. 100 µg anti-NK1.1 (clone PK136, BioXCell) or IgG2a control (Clone C1.18.4, BioXCell) were administered intraperitoneally every sixth day to deplete NK cells. 2.5 mg anti-IFNAR1 (clone MAR1-6A3, Leinco) or IgG1 control (clone HKSP, Leinco) were administered intraperitoneally to block IFNAR1 in SRα-tTA/tet-O-MYC mice bearing overt lymphoma at the same time as the mice were subjected to MYC inactivation with doxycycline treatment. After 4 days, spleen was collected and subjected to flow cytometry for NK and T cell analysis.

Swaminathan S., Hansen A.S., Heftdal L.D., Dhanasekaran R., Deutzmann A., Fernandez W.D., Liefwalker D.F., Horton C., Mosley A., Liebersbach M., Maecker H.T, & Felsher D.W. (2020). MYC functions as a switch for natural killer cell-mediated immune surveillance of lymphoid malignancies. Nature Communications, 11, 2860.

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T Cell and NK Cell Depletion

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To deplete NK cells, CD4⁺ T cells and CD8⁺ T cells, tumour-bearing mice were intraperitoneally injected with anti-NK1.1 (clone PK136, BioXCell), anti-CD4 (clone GK1.5, BioXCell), anti-CD8-α (clone 2.43, BioXCell) or isotype control (RatIgG1,BioXcell) antibodies at an initial dose of 400 mg 1 d before treatment, followed by 200 mg every 3 d. Depletion of CD8⁺ T cells, CD4⁺ T cells and NK cells was confirmed using flow cytometry analysis of peripheral blood mononuclear cells.

Wang F., Su H., Xu D., Dai W., Zhang W., Wang Z., Anderson C.F., Zheng M., Oh R., Wan F, & Cui H. (2020). Tumour sensitization via the extended intratumoural release of a STING agonist and camptothecin from a self-assembled hydrogel. Nature biomedical engineering, 4(11), 1090-1101.

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Targeted Immunomodulation in Lung Cancer

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The anti-NK1.1 depletion experiment was performed in Kras/p53 driven lung cancer model, while IFNγ and CCRL2 blocking in the LG cell line lung metastasis model. Mice were treated at day -1 intraperitoneally with 100 μg of anti-NK1.1 (clone PK136) or isotype control (clone C1.18.4) and anti-CCRL2, generated in the lab (Otero et al., 2010 (link)), or isotype control (clone MPC-11); and 200 μg of anti-IFNγ (clone XMG1.2) or isotype control (IgG1k HRPN) (all from BioXCell). Mice were then treated with 100 μg once (anti-NK1.1) or three times (anti-CCRL2; anti-IFNγ) a week for the entire duration of the experiment.

Del Prete A., Sozio F., Schioppa T., Ponzetta A., Vermi W., Calza S., Bugatti M., Salvi V., Bernardini G., Benvenuti F., Vecchi A., Bottazzi B., Mantovani A, & Sozzani S. (2019). The atypical receptor CCRL2 is essential for NK cell-dependent resistance against lung cancer. Cancer immunology research, 7(11), 1775-1788.

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Therapeutic Anti-GITR and Anti-PD-1 Protocol

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Therapeutic anti-GITR (Clone DTA-1; Catalog#:BE0063), anti-PD-1 (Clone RMP1-14;
Catalog#BE0146), anti-CD4 (Clone GK1.5; Catalog#:BE0003-1), anti-CD8 (Clone 2.43;
Catalog#:BE0061), anti-NK1.1 (Clone PK136; Catalog#:BE0036) and control rat IgG
(Clone 2A3; Catalog#:BE0089) monoclonal antibodies (mAb) were purchased from BioXcell
(West Lebanon, NH). Antibodies used for flow cytometry were purchased from Tianjing
Sungene (eBioscience, San Diego, CA) and eBioscience (San Diego, CA).
H-2Db-restricted mesothelin-derived (MESO406-414: GQKMNAQAI) or control lymphocytic
choriomeningitis virus (LCMV) glycoprotein (GP)-derived (GP33-41: KAVYNFATC) epitope
peptide were synthesized by GenScript (Beijing, China) and more than 95% of purity
were confirmed by HPLC. Peptides were reconstituted in DMSO with final concentration
of 10 mg/mL.

Lu L., Xu X., Zhang B., Zhang R., Ji H, & Wang X. (2014). Combined PD-1 blockade and GITR triggering induce a potent antitumor immunity in murine cancer models and synergizes with chemotherapeutic drugs. Journal of Translational Medicine, 12, 36.

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Adoptive Transfer of Antigen-Specific T Cells

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On day 0, CB6F1 and WT B6 mice were adoptively transferred intravenously with 1 × 10⁶ CD45.1⁺ TEa cells, which were isolated from spleens of CD45.1⁺ TEa mice using the Dynabeads untouched mouse CD4 cells kit (Thermo Fisher Scientific). WT B6 mice were also transplanted with Balb/c tail skins on day 0, whereas CB6F1 mice were intraperitoneally injected with 500 μg anti-NK1.1 (clone PK136, Bio X Cell) on days −3 and 15 to deplete NK cells. The following experiments were then performed: (1) on different days after TEa cell transfer, TEa cell states in peripheral blood and spleens were determined by flow cytometric analysis; (2) on day 6 after TEa-cell transfer, TCR(Vα2⁺ Vβ6⁺)CD45.1⁺CD4⁺ TEa cells were sorted from splenocytes, followed by RNA-seq analysis as described later; and (3) on day 30 after TEa cell transfer, TCR(Vα2⁺ Vβ6⁺)CD45.1⁺CD4⁺ TEa cells were sorted from splenocytes and further adoptively transferred into B6.Rag1^−/− mice. One day later, B6.Rag1^−/− mice were transplanted with Balb/c tail skins to determine the antigraft function of TEa cells.

Zou D., Dai Y., Zhang X., Wang G., Xiao X., Jia P., Li X.C., Guo Z, & Chen W. (2020). T cell exhaustion is associated with antigen abundance and promotes transplant acceptance. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons, 20(9), 2540-2550.

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Selective Depletion of Immune Cells

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CD4⁺ T cells, CD8⁺ T cells, and NK cells were depleted by intraperitoneal (i.p.) injection of 200 μg of anti-CD4 (clone GK1.5), anti-CD8 (clone YTS 169.4), and anti-NK1.1 (clone PK136) antibodies respectively in antibody dilution buffer pH 7.0 (Bio X Cell). Cellular depletion was confirmed by flow cytometry analysis of CD4⁺ T cells, CD8⁺ T cells and NK cells in the blood samples of mice within 24 hours after the first depletion antibody injection. The next day, mice were challenged with 5 × 10⁵ LL/2-tdTomato/Luc cells and another four doses of cellular depletion antibodies were given every four to five days post tumor challenge to deplete newly formed lymphocytes.

Huang L., Bommireddy R., Munoz L.E., Guin R.N., Wei C., Ruggieri A., Menon A.P., Li X., Shanmugam M., Owonikoko T.K., Ramalingam S.S, & Selvaraj P. (2021). Expression of tdTomato and luciferase in a murine lung cancer alters the growth and immune microenvironment of the tumor. PLoS ONE, 16(8), e0254125.

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Induction and Assessment of Active and Passive EAE

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Active EAE was induced by immunization with the myelin oligodendrocyte glycoprotein (MOG) peptide amino acids 35–55 (MEVGWYRSPFSRVVHLYRNGK). 100 μg MOG_35-55 peptide was emulsified in complete Freund’s adjuvant supplemented with M. tuberculosis extract H37Ra (Difco), and injected subcutaneously. Mice received 150 ng pertussis toxin (List Biological Laboratories) intraperitoneally on days 0 and 2. Passive transfer EAE was induced by injecting 2D2 T_H17-polarized cells (as described above) intravenously into recipient mice at 5–7.5 × 10⁶ cells/mouse. For NK depletion experiments, 0.5 mg/dose of purified anti-NK1.1 (clone PK136, BioXCell) was injected intravenously into mice on days −5/−3/−1 prior to 2D2 adoptive transfer, and additionally every 3 days from day 1 post-2D2 transfer. Classical EAE symptoms were scored daily according to standard criteria: 0, asymptomatic; 1, flaccid tail; 2, hind limb weakness and impaired righting ability; 3, hind limb paralysis; 4, front and hind limb paralysis; 5, moribund or death.

Kwong B., Rua R., Gao Y., Flickinger J J.r., Wang Y., Kruhlak M.J., Zhu J., Vivier E., McGavern D.B, & Lazarevic V. (2017). T-bet-dependent NKp46+ innate lymphoid cells regulate the onset of TH17-induced neuroinflammation. Nature immunology, 18(10), 1117-1127.

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