Total RNA was purified from human tumor-infiltrating TREG (LIN−CD45+CD3+CD4+CXCR5−CD127−CD25+) and TFR (LIN−CD45+CD3+CD4+CXCR5+GITR+) cells using a miRNeasy kit (Qiagen) and quantified as described previously47 (link),49 (link). Cells from mice immunized with either OVA in Complete Freund’s adjuvant (InvivoGen), OVA in monophosphoryl lipid A (InvivoGen) or mock PBS—TEFF (CD19−CD45+CD3+CD4+CXCR5−GITR−CD25−CD62L−CD44+), TREG (CD19−CD45+CD3+CD4+CXCR5−GITR+CD25+), TFH (CD19−CD45+CD3+CD4+CXCR5+GITR−) and TFR (CD19−CD45+CD3+CD4+CXCR5+GITR+)—were sorted and RNA was purified as described above. RNA-seq libraries were prepared using Smart-seq2 protocol and sequenced on an Illumina platform, as previously described50 (link). Quality-control steps were applied as previously described47 (link). Samples that failed the quality controls or that had a low number of cells were excluded from further sequencing and analysis.
Complete freund s adjuvant
Manufactured by InvivoGen
Sourced in United States
Complete Freund's adjuvant is a laboratory reagent used to enhance the immune response in experimental animals. It contains heat-killed and dried mycobacteria suspended in a mineral oil and emulsifier. The adjuvant is used to potentiate the immune response to an antigen during vaccine and immunological studies.
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Lab products found in correlation
Mirneasy kit, by Qiagen (1 mentions)
Methotrexate, by Merck Group (1 mentions)
Glutathione reductase, by Merck Group (1 mentions)
Nitrotetrazolium blue chloride nbt, by Merck Group (1 mentions)
Silibinin, by Merck Group (1 mentions)
Glutathione (gsh), by Merck Group (1 mentions)
Incomplete freund s adjuvant, by InvivoGen (1 mentions)
C57bl 6j mice, by Jackson ImmunoResearch (1 mentions)
Polyvinyl alcohol pva 1500, by Duksan Pure Chemicals (1 mentions)
Ovalbumin (ova), by Merck Group (1 mentions)
Intracellular fixation and permeabilization buffer set, by Thermo Fisher Scientific (1 mentions)
Brefeldin a, by Thermo Fisher Scientific (1 mentions)
Pertussis toxin, by Thermo Fisher Scientific (1 mentions)
Mog35 55, by GenScript (1 mentions)
M tuberculosis, by BD (1 mentions)
7 protocols using complete freund s adjuvant
1
Purification and Transcriptome Profiling of Regulatory T-Cell Subsets
2
Freund's Adjuvant-Induced Oxidative Stress Assay
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Complete Freund’s adjuvant was purchased from InvivoGen, San Diego, CA, USA (vac-cfa-10). Methotrexate (≥98% (HPLC), A6770); silibinin (containing both A and B diastereomers, ≥98% (HPLC), S0417); DTNB (≥98%, D-8130); NADPH (≥97% (HPLC), N-7505); glutathione (GSH ≥ 98.0%, G4251); glutathione reductase; polyvinylpolypyrrolidone (PVPP, 77627); and nitrotetrazolium bluechloride (NBT, ≥90.0% (HPLC), 298-83-9) were purchased from (Sigma-Aldrich Co, St. Louis, MA, USA). Alanine aminotransferase (ALT) kits were purchased from Analyticon ® Biotechnologies AG, Frankfurt, Germany. Aspartate aminotransferase (AST) kits were purchased from Spinreact, S.A./S.A. U Ctra. Santa Coloma, Gerona, Spain. Trichloroacetic (TCA); thiobarbituric acid (TBA); hydrogen peroxide, (H2O2 30% w/v); L-methionine (149 g/mol); riboflavin (376 g/mol); potassium dihydrogen orthophosphate (KH2PO4); dipotassium hydrogen orthophosphate (K2HPO4); and EDTA sodium salt were made available by the Beirut Arab University.
3
Neoantigen Vaccine and Checkpoint Inhibitor Study
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Mice were prophylactically vaccinated subcutaneously with 100 ug of mRiok1 peptide in 100 uL PBS in combination with 1:1 volume of Complete Freund's Adjuvant (InvivoGen) (day -12). Blood was collected on day 7 and fluorescence-activated cell sorting (FACS) stained to confirm the presence of mRiok1-tet+ CD8 T cells (day -5). Mice were boosted with another shot of neoantigen vaccine, and tumor cells were injected 5 d after (day 0). When applicable, anti–PD-1 and/or anti–CTLA-4 treatment was given as described in the Antibody Treatment section. As controls, mice were immunized with either PBS or the mRiok1 peptide alone.
4
Vaccine Emulsion Preparation and Injection
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The first vaccine emulsion was prepared by mixing Complete Freund’s Adjuvant (Invivogen) and R-P4 (20 μM dissolved in sterile PBS) at a ratio of 1:1. For the first vaccination, 100 μL of the emulsion was injected (IP) into male (n = 12) and female (n = 12) C57BL6/J mice (obtained from Jackson Laboratories). Fourteen days after initial vaccination, mice were injected with 100 μL of freshly prepared vaccine emulsion in InComplete Freund’s Adjuvant (Invivogen). Adjuvant-only emulsions were made by mixing the appropriate adjuvant in sterile PBS at a ratio of 1:1. Adjuvant-only control mice were injected (IP) in male (n = 12) and female (n = 12) mice on the same schedule as analog-vaccinated mice.
5
Curcumin-Loaded PLGA Nanoparticles
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Poly(D,L-lactic-co-glycolic) acid (PLGA, MW 7000–17,000, cas#26780-50-7) from Sigma-Aldrich®, St. Louis, MO, USA; polyvinyl alcohol (PVA 1500, MW 44.053 g/mol) from Duksan® Pure Chemicals, Seoul, Korea; curcumin (Cur) from Spectrum®, Shanghai, China; meloxicam (Mlx) from Sigma-Aldrich®, USA; and Complete Freund’s adjuvant from Invivogen®, San Diego, CA, USA, France were purchased. All other reagents and solvents of highest purity were used. These chemicals were purchased under the Higher Education Commission (H.E.C.), Islamabad, Pakistan, grant number 7510.
6
Adoptive Transfer and Immunization of OT-1 T Cells
7
EAE Induction and Clinical Scoring Protocol
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EAE was induced 4 weeks after the tamoxifen treatment, by giving the mice a 200 µl subcutaneous injection containing 200 µg of myelin oligodendrocyte glycoprotein (MOG)35-55 (GenScript, New Jersey, USA) in a complete Freund´s adjuvant (InvivoGen, San Diego, CA, USA) containing 300 µg of M. tuberculosis (BD, Sparks, MD, USA). Simultaneously and 48 h later, mice were injected intraperitoneally with 300 ng of pertussis toxin (Invitrogen, Carlsbad, CA, USA). Clinical manifestations of EAE were scored as follows: 0, no clinical manifestations; 1, tail paralysis; 2, hind limb paresis; 3, hind limb paralysis; 4, partial-fore limb paralysis. Intermediate 0.5 scores were assigned if the animals displayed the symptoms between the two scores. Tissue for histological and molecular biological analyses was taken from mice that had reached a certain score and remained at that score for 3 consecutive days (considered as peak of the disease). If no symptoms developed, animals were followed for a total of 26 days before they were killed.
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