Antibodies used include: Ii (In-1, 1:100), H2-M (2E5A, 1:50), Rat IgG1 (R3-34, 1:50; all from BD Biosciences); podoplanin (8.1.1, BioLegend, 1:500); CLIP (15G4, Santa Cruz, 1:5); H2-O (ref. 69 (link); Mags.Ob3, Lisa Denzin, 1:400); 10.1.1 (UVA lymphocyte culture centre, 1:1,000); CD45 (30-F11, 1:1,000), CD31 (390, 1:1,000), MHC-II (M5/114.15.2, 1:50 (Fig. 1 ) to 1:500 (Fig. 6 )), CD8 (53-6.7, 1:1,000), CD4 (GK1.5, 1:1,000), Thy1.1 (HIS51, 1:1,000), Thy1.2 (53-2.1, 1:1,000), CD45.1 (A20, 1:500), CD11c (N418, 1:500), CD11b (M1/70, 1:500), CD69 (H1.2F3, 1:500), CD62L (MEL-14, 1:1,000), CD44 (IM7, 1:1,000), CD25 (PC61.5, 1:500), Y-AE (eBioY-Ae, 1:200), PD-1 (RMP1-30, 1:100), LAG-3 (eBioC9B7W, 1:100), Rat IgG2b (eB149/10H5; all from eBioscience). Intracellular staining for PD-1, LAG-3, Ii, H2-M and H2-O was done using the Cytofix/Cytoperm kit (BD Bioscience), and Ki67 (SolA15, 1:500) and FoxP3 (FJK-16s, 1:100) were stained using Treg permeabilization buffers (eBioscience). Annexin V (1:20) was stained using the eBioscience kit. 4,6-Diamidino-2-phenylindole (DAPI; Sigma) or live/dead aqua (Invitrogen, 1:200) were used to distinguish live cells. Cells were acquired on a FACSCanto II (BD Biosciences) and data analysed using FlowJo (Tree Star).
Rmp1 30
Manufactured by Thermo Fisher Scientific
Sourced in United States
The RMP1-30 is a centrifugal mixer designed for laboratory use. It features a compact, benchtop design and is capable of mixing a variety of sample types at speeds up to 3,000 rpm.
Automatically generated - may contain errors
Lab products found in correlation
Attune nxt, by Thermo Fisher Scientific (2 mentions)
Annexin 5, by Thermo Fisher Scientific (1 mentions)
Rat igg2b eb149 10h5, by Thermo Fisher Scientific (1 mentions)
Treg permeabilization buffers, by Thermo Fisher Scientific (1 mentions)
Clip 15g4, by Santa Cruz Biotechnology (1 mentions)
Ebioy ae, by Thermo Fisher Scientific (1 mentions)
Cytofix cytoperm kit, by BD (1 mentions)
Anti nk1.1 pk136, by BioLegend (1 mentions)
Anti cd8 53 6.7, by BioLegend (1 mentions)
Rm4 4, by BioLegend (1 mentions)
Soluble anti cd28 mab, by BioLegend (1 mentions)
Anti cd3ε mab, by BioLegend (1 mentions)
Facsdiva, by BD (1 mentions)
Facsaria 2, by BD (1 mentions)
4 protocols using rmp1 30
1
Multicolor Flow Cytometry Panel
2
Combination Therapy for Tumor Regression
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For comparing the efficacy of singular or combined therapies consisting of an adoptive transfer of ex vivo TKD/IL-2-stimulated mouse/human NK cells and mouse/human anti-PD-1 immune checkpoint inhibitor antibody (RMP1-30, eBioscience, Frankfurt/Main, Germany) animals with comparable tumor sizes (according to MRI volumometrics) were randomly divided into 5 groups (8 animals per group): Animals of the control groups were injected either with 100 μl PBS (iv) or with 250 μg isotype-matched IgG antibody (ip) on days 6, 9, 12 and 15. Animals of the treatment groups were iv injected either with NK cells (6 × 106 in 100 μl PBL) on days 6, 9, and 12 and/or ip injected with anti-PD-1 antibody on days 6 (500 μg), 9 (250 μg), 12 (250 μg), and 15 (250 μg) in a volume of 500 μl PBS.
3
Quantifying Tumor-Infiltrating Immune Cells
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Animals were anesthetized by ip injection of 150–200 mg/kg pentobarbital. After perfusion with 100 ml saline/4% paraformaldehyde, whole brains were removed and tumor volumes were assessed. Tissue was fixed in 4% paraformaldehyde/30% sucrose, embedded in Tissue-Tek® and blocks were cut into serial sections (5–7 μm). CD8+ T cells, NK1.1+ cells and PD-1+ lymphocytes were stained on IHC sections using anti-CD8 (53-6.7, Biolegend, San Diego, CA, USA), anti-NK1.1 (PK136, Biolegend, San Diego, CA, USA) and anti-PD-1 (RMP1-30, eBioscience, Frankfurt/Main, Germany) antibodies according to an established protocol. Tumor-infiltrating CD8+ T cells, NK1.1 cells and PD-1+ cells were counted in 3 fields of views by two independent researchers.
4
Isolation and Functional Analysis of DMBA-Induced Liver Immune Cells
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Immune cells from livers of DMBA-treated HFD-fed WT mice were labeled with antibodies to mouse CD8a (53-6.7, BioLegend), CD4 (RM4-4, BioLegend), and PD-1 (RMP1-30, eBioscience), and PD-1 -or PD-1 + CD8 T cells were sorted using FACSDiva and FACSAria II (BD Biosciences). Purified PD-1 -or PD-1 + CD8 T cells (0.25 × 105) were seeded with pre-coated anti-CD3ε mAb (5 μg/mL, BioLegend) and soluble anti-CD28 mAb (2.5 μg/mL BioLegend) for 3 days at 37 °C under 5% CO2 in 96-well flatbottomed plates in a volume of 0.2 ml RPMI containing 10% FBS and 1% streptomycin plus penicillin. After incubation, the culture supernatant was collected, and cytokine concentration was measured by an Enzyme-Linked Immunosorbent Assay (ELISA) development kit for mouse tumor necrotic factor (TNF), interferon-gamma (IFN-γ), and interleukin-2 (IL-2) (eBioscience). Cultured cells were fixed, permeabilized, and stained for Ki-67 (BioLegend). Data were acquired by Attune Nxt (Thermo Fisher) and analyzed with FlowJo (Tree Star). Detailed antibody information is shown in Table S1.
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