This study was approved by the Mayo IRB. Blood samples were obtained from normal donors (n=15) and ovarian cancer patients (n=11). Tumor (n=15) and ascites (n=11) samples were obtained from ovarian cancer patients. Tumor specimens were minced into <1 mm3 pieces, isolated into single cells using gentleMACS™ tissue dissociator (Miltenyi, San Diego, CA), passed through a 40μm filter and processed by ficoll gradient as previously described (10 (link), 25 (link)). CD1c+ cells used in in vitro assays were isolated from the samples using human CD1c+ (BDCA-1+) DC isolation kit (Miltenyi). Blockade of human PD-1 was accomplished using a purified PD-1 antibody from BioLegend (Cat. # 329912, San Diego, CA).
Pd 1 antibody
Manufactured by BioLegend
Sourced in United States
The PD-1 antibody is a laboratory reagent used in immunological research. It binds to the PD-1 protein, which is expressed on the surface of certain immune cells. The PD-1 antibody can be used to investigate the role of the PD-1 pathway in immune responses and its potential implications in areas such as cancer biology and autoimmune disorders.
Automatically generated - may contain errors
Lab products found in correlation
Anti human lag3, by BioLegend (2 mentions)
Sytox blue, by Thermo Fisher Scientific (2 mentions)
Propidium iodide, by BD (2 mentions)
Binding buffer, by BD (2 mentions)
Gentlemacs tissue dissociator, by Miltenyi Biotec (1 mentions)
4 6 diamidino 2 phenylindole dapi, by Boster Bio (1 mentions)
Cell counting kit 8 cck 8, by Dojindo Laboratories (1 mentions)
N 3 dimethylaminopropyl n ethylcarbodiimide hydrochloride edc, by Merck Group (1 mentions)
N hydroxysuccinimide, by Merck Group (1 mentions)
Tepp 46, by MedChemExpress (1 mentions)
Annexin 5, by Thermo Fisher Scientific (1 mentions)
6 protocols using pd 1 antibody
1
Ovarian Cancer Immune Profiling
2
PD-1 Expression on HMC-1 Cells
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HMC-1 cells were fixed with 4% paraformaldehyde for half an hour. The cells were washed with PBS three times after removal from 4% paraformaldehyde. Under dark conditions, the cells were incubated with antibodies for half an hour. One group was incubated with PD-1 antibody (1:100, #135225, Biolegend), and the other group was incubated with isotype control antibody (1:100, #400546, Biolegend). The cells were washed with PBS three times and then analyzed.
3
Exosome Regulation of T Cell PD1 Expression
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Jurkat T cells were seeded onto 12-well dishes, and 500 μg of exosomes were added into each well. After incubation for 24 h, the Jurkat T cells were collected for qRT-PCR or flow cytometry analysis. The Jurkat T cells were stained with the PD1 antibody (BioLegend, Clone EH12.2H7), and the expression of PD1 was determined by flow cytometry. Data were analyzed with FlowJo-10 software.
4
Synthesis of PLGA-PEG Nanoparticles
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PLGA-COOH (50:50, 30kDa) including a premixed content of ~3.4kDa polyethylene glycol (PEG) was purchased from Xi’anRuiXi Co., Ltd (Xi’an, China). Polyvinyl alcohol (PVA, 26 kDa), 1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI), 2- (N-morpholino) ethanesulfonic acid (MES monohydrate), N- (3-dimethyl-ami-nopropyl)-N’-ethylcarbodiimide hydrochloride (EDC) and N-hydroxy-succinimide (NHS) were purchased from Sigma-Aldrich Co., Ltd (St Louis, MO, USA). Dimethyl sulfoxide (DMSO) was purchased from Solarbio Ltd (Beijing, China). Tepp-46 was obtained from MedChemExpress (New Jersey, USA). PD-1 antibody was purchased from Biolegend (114114, San Diego, USA). Human interphotoreceptor-retinoid-binding protein (IRBP651-670, LAQGAYRTAVDLESLASQLT) peptide was produced by Sangon Biochem Ltd (Shanghai, China). 4’,6-diamidino-2-phenylindole (DAPI) was purchased from Boster Biological Technology Co., Ltd (California, USA). Cell counting kit-8 (CCK-8) was purchased from Dojindo (Kyushu, Japan).
5
Characterizing Regulatory T Cells in Autoimmune Diseases
6
Phenotyping of Activated Tregs
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FACS-sorted memory Tregs from patients with dnAIH, AIH, and LTC were resuspended in PBS at a concentration of 1 X 10 6 cells/ml and subjected to culture in the presence of rIL-2, over 24-48-hours. Tregs were then washed twice with cold PBS and re-suspended in 1X binding buffer (BD Pharmingen, #556547) at a concentration of 1 X 10 6 cells/ml. 100 μl (1 X 10 5 cells) of re-suspended cells were transferred to a 5 ml V bottom tube, 5 μl of Annexin V and 1 μl of Sytox Blue (Thermo Fisher Scientific #S34857) [or 5 μl of Propidium Iodide (BD Pharmingen, # 556547)], and 1 μl each of anti-human LAG3 (Biolegend #369309), and PD1 antibody (Biolegend #367427) were added and incubated in the dark for 15-minutes at room temperature. Following incubation, 400 μl of 1X binding buffer were added and samples were analyzed by flow cytometry.
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