Lymphoma cells expressing MHC class-I (RMA) were labeled with a low CFSE concentration (1 μM), whereas those with defective expression of MHC class-I (RMA-s) were labeled with a high CFSE concentration (4 μM). The cells were mixed in a 1:1 ratio (1 × 106 cells per each cell type) and injected i.p. into C57BL/6 mice. To deplete the NK cells, i.p. injections of rabbit anti-asialo-GM1 (10 μl; Cedarlane Laboratories Ltd) or control rabbit serum (Sigma-Aldrich) were performed for 3 consecutive days before an injection of CFSE-stained RMA and RMA-s cells at a ratio of 1:1. Rejection of NK cell-sensitive RMA-s relative to NK cell-resistant RMA cells in the peritoneal cavity was measured using flow cytometry and calculated as follows: 1 – ([CFSElow/CFSEhigh]input/[CFSElow/CFSEhigh]output) × 100%.
Anti asialo gm1
Manufactured by Cedarlane
Sourced in Canada
Anti-asialo-GM1 is a laboratory reagent used to deplete natural killer (NK) cells in experimental settings. It functions by selectively binding to and removing asialo-GM1-expressing cells, which are primarily NK cells, from a sample or cell population.
Automatically generated - may contain errors
Lab products found in correlation
Control rabbit serum, by Merck Group (1 mentions)
Chrompure rat igg, by Jackson ImmunoResearch (1 mentions)
Anti nk1.1 clone pk136, by BioXCell (1 mentions)
Anti cd4 clone gk1, by BioXCell (1 mentions)
Anti cd8 clone 2, by BioXCell (1 mentions)
Clodronate liposome, by Liposoma (1 mentions)
Anti cd4, by BioXCell (1 mentions)
Anti ly6g, by BioXCell (1 mentions)
6 protocols using anti asialo gm1
1
MHC Class-I NK Cell Rejection Assay
2
Immune Subset Depletion and Checkpoint Blockade
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To deplete specific immune subsets, mice were treated with intraperitoneal (i.p.) injections (0.1 mg per mouse) of anti-CD8 (Lyt 2.43, BioXCell), anti-CD4 (GK1.5, BioXCell), anti-NK (anti-asialo-GM-1, Cedarlane), and IgG control (ChromPure Rat IgG, Jackson ImmunoResearch) at day 4 after tumor implantation and then weekly thereafter. Fluorescence-activated cell sorting analysis of spleens and lymph nodes confirmed subset specific depletions. For immune checkpoint blockade, mice were treated intravenously with anti-PD1 (0.25 mg; catalog no. BE0146; Bio X Cell), anti-CTLA-4 (0.1 mg; catalog no. BE0164; Bio X Cell), anti-asialo GM1 (0.1 mg; catalog no. CL8955; Cedarlane), or isotype control rat IgG (catalog no. 012-000-003; Jackson ImmunoResearch) antibody at times described in each experiment.
3
T-cell and NK-cell Depletion Protocol
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To deplete CD4+ or CD8+ T-cell populations, mice were given 4 daily i.p. injections of 100 μg GK1.5 or 2.43 antibodies, respectively, the week before tumor implantation. Mice were then given additional weekly i.p. injections throughout the study to maintain these low T-cell levels. NK cells were depleted by injecting mice once per week with 25μl rabbit anti-asialo-GM1 (Cedarlane Laboratories), beginning the week before tumor cell injection and continuing through the end of the study.
4
Selective Depletion of T Cell Subsets
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In order to selectively deplete CD4+ or CD8+ T cells in vivo, mice were injected with GK1.5 or 2.43 antibody, respectively. Mice were injected i.p. with 100μg of the appropriate antibody each day for 4 consecutive days, beginning 1 week prior to tumor implantation. Mice were given additional antibody injections each week throughout the study in order to maintain low T cell numbers. NK cells were depleted by injecting mice weekly with 25μl rabbit anti-asialo-GM1 (Cedarlane Laboratories, Burlington, Ontario, Canada), beginning 1 week prior to tumor implantation and continuing throughout the study.
5
In Vivo Immune Cell Depletion
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For in vivo depletion studies, mice were administered four daily i.p injections of depleting antibodies prior to tumor instillation, followed by weekly i.p injections of depleting agents after initiation of tumor. CD4+ T cell depletion was accomplished via administration of 100 µg/injection of anti-CD4 clone GK1.5 (BioXcell, Branford, CT). CD8+ T cell depletion was accomplished via administration of 100 μg/injection of anti-CD8 clone 2.43 (BioXcell, Branford, CT). NK cell depletion was accomplished via administration of 25 µL/injection of anti-NK1.1 clone PK136 (BioXcell, Branford, CT) and 25 µg/injection of polyclonal anti-asialo GM1 (Cedarlane Laboratories, Burlington, Canada).
6
Immune Cell Depletion Protocol
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Depletion of neutrophils and T-cell subsets was achieved by i.p. injection of 200 μg antibody (BioXCell, Lebanon, NH, USA) every 3 days, beginning 3 days prior to cell transplantation, using the following antibodies: anti-Ly6G (clone 1A8), anti-CD8α (clone 2.43), anti-CD4 (clone GK1.5), and anti-Vγ2 TCR (clone UC3-10A6). For macrophage and NK-cell depletion, 200 μg of Clodronate liposomes (Liposoma BV, Amsterdam, Netherlands) and anti-asialo GM1 (Cedarlane, Burlington, ON, Canada), respectively, was injected 24 hours before cell transplantation and 50 μg every 3 days after. Depletion of each cell type was confirmed by flow cytometry of peripheral blood mononuclear cell (PBMC) before injecting each new dose of antibodies (data not shown). Antibodies used for depletion were from different clones than those used in flow cytometry to confirm the depletion.
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