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37 protocols using rituximab

1

Establishment of Ovarian Cancer PDX Models

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Fresh human tumor tissues from consenting patients with ovarian cancer were collected at the time of primary debulking surgery and coded with a patient heterotransplant (PH) number in accordance with the Mayo Clinic Institutional Review Board and the Health Insurance Portability and Accountability Act regulations. All animal procedures were approved by the Mayo Clinic Institutional Animal Care and Use Committee (IACUC). Tumors were established by IP injection into female SCID beige mice (C.B.-17/IcrHsd- Prkdcscid Lystbg; ENVIGO) as previously described89 (link). Briefly, minced patient tumor in McCoy’s 5a medium was supplemented with rituximab (10 mg/kg, Genentech, Inc.) to prevent lymphoma development179 (link) in ∼0.3 mL of total volume for each injection. After engraftment, PDX tumors were expanded into additional mice prior to cryogenic preservation for future experiments89 (link). The minimal information standard for PDX models is provided in the following table:
048
GenderF
Age60
DiagnosisOvarian Cancer
ConsentAcademic
Primary TissueOvary
Collection SitePrimary
Specimen collectedOvary
HistologySerous
GradeHigh
StageIIIC
MarkersN/A
TreatmentNaive
Mouse StrainSCID-bg
Mouse HumanizedNo
PreparationSolid Tumor
Injection siteIP
CharacterizationHistology
Negative murine/EBVYes
PassageP7
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2

Particle Size and Aggregation of ABX and Rituximab

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ABX (Celgene, Summit, NJ) and rituximab (Genentech, San Francisco, CA) were mixed at 10 mg/ml and 4 mg/ml, respectively in 0.9% saline and incubated for 30 minutes at room temperature. For the peptide competition assay, 10 mg/ml of ABX was incubated for 30 minutes with 4 mg/ml of rituximab and a 10 molar excess of a control peptide, HSA Peptide 40, or no peptide. We determined particle size and number by Malvern Nanosight (Malvern, Worcestshire, UK), which uses light scattering and Brownian motion to obtain particle size and concentration.
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3

Antibody Characterization and Modification

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2-bromoethylamine hydrobromide, TRIS hydrochloride, trypsin, α-cyano-4-hydroxycinnamic acid (CHCA), acetylated cysteine (Ac-C), and acetic anhydride were purchased from Sigma (St. Louis, MO, USA). Glu-C was from Promega (Madison, WI, USA). Potassium phosphate was acquired from JT Baker (Phillipsburg, NJ, USA) and ammonium bicarbonate was from Mallinckrodt Chemicals (Phillipsburg, NJ, USA). Concentrated sodium hydroxide was obtained from VWR Analytical (Radnor, PA, USA) and dithiothreitol was purchased from Bio-Rad (Hercules, CA, USA). Genentech (South San Francisco, CA, USA) provided the antibody, rituximab. Fmoc-Arg(Ptf) Wang resin and Fmoc-Cys(Trt)-OH were from Midwest Biotech (Fishers, IN, USA).
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4

Evaluating Anti-Cancer Drug Synergies

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All chemicals were purchased from Sigma-Aldrich (St Louis, MO, USA) or Fisher Scientific (Pittsburgh, PA, USA) unless specified otherwise. Ficoll-Paque Plus was obtained from Amersham Biosciences (Piscataway, NJ, USA). The media for cell culture were purchased from GIBCO (Grand Island, NY, USA). The anti-CD-20 APC antibody was obtained from eBiosciences (San Diego, CA, USA). CellTiter 96® Non-Radioactive Cell Proliferation Assay kit (MTT) was purchased from Promega (Madison, WI, USA). Fludarabine was obtained from Teva Parenteral Medicines, Inc (Irvine, CA, USA). Rituximab was obtained from Genentech (South San Francisco, CA, USA). BIBB-515 and YM-53601 were purchased from Cayman Chemical Company (Ann Arbor, MI, USA). TAK-475 was obtained from Chemzon Scientific (Montreal, Quebec, Canada).
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5

Comprehensive Immunophenotyping of haNK Cells

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The cetuximab (Bristol-Myers Squibb, New York, NY), trastuzumab (Genentech, San Francisco, CA), pertuzumab (Genentech) and isotype (rituximab, Genentech) antibodies were purchased from the National Institutes of Health Pharmacy. Flow cytometry of haNK cells was performed on a BD LSRII flow cytometer (BD Biosciences, San Jose, CA) and analyzed in FlowJo 9.9 (TreeStar Inc., Ashland, OR). Flow cytometry of tumor cells was performed on a BD FACSVerse flow cytometer (BD Biosciences) and analyzed in BD FACSuite (BD Biosciences). Staining of haNK cells was performed with four panels, and the antibodies used were: Anti-CD56-PErCP-Cy5.5, anti-CD16-APC-Cy7, anti-NKG2D-APC, anti-NKG2D-FITC, anti-CD107a-PE-Cy7, anti-GranzymeB-FITC, anti-perforin-APC, anti-NKp46-PE-Cy7, anti-NKp30-APC, anti-NKp44-PE, anti-2B4-FITC, anti-CD158a-PE, anti-CD158d-PE, anti-CD158j-APC, anti-PD-1-PE-Cy7, anti-PD-L1-APC, and anti-MICA-PE; all were obtained from BioLegend (San Diego, CA). Anti-CD56-PE, anti-CD16-PE-Cy7, CD11a-PE, Ki67-FITC, anti-HLA-ABC-FITC and anti-4-1BB-BV421 were obtained from BD Biosciences. In controlled experiments to detect CD16 on haNK cells, the anti-CD16 MAb was CD16-FITC (BD Biosciences) and the isotype control antibody was MIgG1k-FITC (BD Biosciences).
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6

CD20-Targeted Photosensitizer Conjugation

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Water soluble, silica-phthalocyanine derivative, IRDye 700DX NHS ester was obtained from LI-COR Biosciences (Lincoln, NE, USA). Rituximab, a chimeric (mouse/human) monoclonal antibody (mAb) directed against CD20 was purchased from Genentech (South San Francisco, CA, USA). All other chemicals were of reagent grade.
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7

Cultivation and Characterization of Diverse Cell Lines

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K562 (Chronic Myelogenous Leukemia), Raji (Burkitt’s lymphoma), HEL 92.1.7 (Erythroleukemia) and NK92MI cell lines were obtained from ATCC and maintained as per the instructions provided. Nalm6 (Acute Lymphoblastic Leukemia), BC-1 (Primary effusion lymphoma), and KG-1 (Acute myelogenous leukemia) cell lines were kindly provided by Drs. Markus Muschen, Jae Jung, and Alan Epstein, respectively. 293FT cells were obtained from Invitrogen (ThermoFisher Scientific) and cultured as recommended. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll gradient method from platelet depleted donor cells obtained from a Blood Bank. PBMCs were subsequently used to isolate T cells using CD3 magnetic microbeads (Miltenyi Biotech) following the manufacturer’s instructions. T cells were cultured in XVIVO-15 (Lonza) medium supplemented with 100 IU/ml recombinant human IL2 and 30 ng/ml soluble antibodies to human CD3 and CD28. All the cells were cultured at 37 °C, in a 5% CO2 humidified incubator. Blinatumomab was obtained from Amgen, Rituximab was obtained from Genentech. Digitonin, and Polybrene were from Sigma. Coelenterazine was purchased from Nanolight technology. Calcein AM fluorescent dye was obtained from BD biosciences.
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8

Isolation and Characterization of Antibodies

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Rituximab (Genentech, San Francisco, CA) was acquired through a drug exchange program at Duke University Medical Center, Department of Hematology and Oncology. Human CFH mAb 7968 was generated by cloning and expressing antibody genes derived from a single B cell as previously described [12 (link)]. The IgG1subclass-matched negative control antibody 7B2 recognizes gp41, an HIV-1 envelope glycoprotein, and was a gift of Dr. H. Liao of the Duke Human Vaccine Institute. The rat anti-human CD59 mAb YTH53.1 was obtained from Santa Cruz Biotechnology (Dallas, TX). The FITC mouse anti-human CD20 mAb 2H7 was obtained from BD Biosciences (San Jose, CA).
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9

Antibody-coated Bead Preparation for Protein Selections

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Streptavidin-coated magnetic beads (NEB) were used for selections/staining for streptavidin-specific DeNAno with no modifications. For rituximab and bevacizumab selections/staining, coated beads were made as follows: 6 μm polystyrene beads (Polysciences, Warrington, PA, USA) were washed with 20 mM sodium phosphate buffer pH 7.5 (Boston BioProducts, Ashland, MA, USA), then coated with 100 μg/ml rituximab (Genentech, South San Francisco, CA, USA), bevacizumab (Genentech), or polyclonal human IgG (Thermo Fisher Scientific, Waltham, MA, USA) diluted in 20 mM sodium phosphate pH 7.5. Beads and antibody were incubated for 2 h at room temperature (RT) or overnight at 4°C. Non-adsorbed antibody was removed with 3 × 1 ml washes with 20 mM sodium phosphate pH 7.5. Finally, beads were resuspended in phosphate bufferedsaline (PBS) (without calcium and magnesium, Mediatech, Manassas, VA, USA) supplemented with 0.02% NaN3 (Ricca Chemical Company, Arlington, TX, USA).
In one control experiment, streptavidin polymethyl methacrylate (PMMA) (Sapidyne Instruments, Boise, ID, USA) and streptavidin sepharose (GE Healthcare Life Sciences, Piscataway, NJ, USA) were used in place of streptavidin magnetic beads.
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10

Assessing Natural Killer Cell Cytotoxicity

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Two assays of NK cell function were performed. In the first test, the NK cell response to missing-self was assayed after challenge and culture of NK cells with class I-deficient K562 cells. The second test measured the ADCC response of NK cells after challenge and culture with Raji cells coated with anti-CD20 antibody. PBMC (5 × 105) were mixed with K562 or Raji cells at a ratio of 10:1 in V-bottom 96-well plates, centrifuged at 1,000 rpm for 3 minutes and incubated at 37°C for 5 hours. Brefeldin A and monensin (both from BD Biosciences) were added to cultures after 1 hour. For K562 stimulation, PBMC cultured in complete medium and nothing else were used as a negative control. For ADCC assays, 2 × 106/ml Raji cells were pre-coated with either Rituximab (Genentech) at 10 µg/ml or with murine IgG at 10 µg/ml for 30 minutes. Raji cells were washed in RPMI/10% FBS and then mixed with PBMC.
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