C57BL/6 wild-type were bred at our animal facility. Gal-1 knock-out (Lgals1−/−) mice were a gift from Dr. Françoise Poirier (Institut Jacques Monod, Université Paris Diderot, Paris, France). Both strains were kept in specific pathogen-free conditions and water was supplied ad libitum. All animal procedures were approved by the ethical committee. Ethical committee name: Commission Ethique Animale—Université de Liège; approval code: # 14-1635 (given on 24 December 2014). Prior to bone marrow transplantation, eight to twelve-week mice were irradiated at 6Gy. Two hours post-irradiation, 2 × 106 bone marrow cells from syngeneic mice were injected via tail vein injection. Hematopoietic stem cells from gal-1−/− mice were injected in recipient gal-1−/− and hematopoietic stem cells from WT mice injected in WT mice. 24 h after transplantation, 2 × 106 5TGM.1 GFP+ cells were inoculated intravenously. During the experiments with bisphosphonates, mice received either PBS (control group; Lonza) or 1.5 mg/kg pamidronate (Sigma-Aldrich) subcutaneously once per week. Bone marrow infiltration of MM cells in mice was determined by FACS detection of GFP+ cells on a FACSCantoII flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). All experimental procedures were approved by the University of Liege (Liège, Belgium) Ethical Committee.
Pamidronate
Manufactured by Merck Group
Sourced in United States
Pamidronate is a bisphosphonate compound used as a laboratory reagent. It is primarily utilized in research applications to inhibit bone resorption and for the treatment of various bone-related disorders.
Automatically generated - may contain errors
Lab products found in correlation
Etidronate, by Merck Group (2 mentions)
Imidodiphosphate, by Merck Group (2 mentions)
32p ppi, by PerkinElmer (2 mentions)
Ddctp, by GE Healthcare (2 mentions)
Chlodronate, by Merck Group (2 mentions)
3 azido 2 3 dideoxythymidine triphosphate azttp, by TriLink (2 mentions)
Arabinofuranosylcytosine triphosphate aractp, by TriLink (2 mentions)
Gemcitabine dfdctp, by Jena Biosciences (2 mentions)
α 35s datp, by PerkinElmer (2 mentions)
Polyethyleneimine pei cellulose thin layer chromatography plates, by Merck Group (2 mentions)
α 32p dctp, by PerkinElmer (2 mentions)
Recombinant human alpl, by R&D Systems (2 mentions)
Shrimp hepatopancreas alp, by New England Biolabs (2 mentions)
P nitrophenyl phosphate kits, by New England Biolabs (2 mentions)
Disodium α glycerophosphate hydrate 1 m of αgp glycophos, by Fresenius (2 mentions)
Alizarin red s solution, by Merck Group (2 mentions)
α mem, by Thermo Fisher Scientific (2 mentions)
Dexamethasone (dex), by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Atlanta Biologicals (2 mentions)
Facscanto 2 flow cytometer, by BD (1 mentions)
9 protocols using pamidronate
1
Gal-1 Knockout Bone Marrow Transplant
2
Human pol β purification and assay
3
Chronic Pamidronate Treatment in OVX Mice
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At 72 h after the last acute treatment, mice (OVX, n = 22; sham-operated, n = 28; 6 months after surgery) were randomly reassigned to receive chronic treatment with either pamidronate (0.25 mg/kg, i.p., Sigma-Aldrich, St. Louis, MO) or a saline vehicle control. The drug or saline was administered 5 days/week for 5 weeks. pamidronate belongs to the bisphosphonate class of medications commonly administered to patients with osteoporosis or bone metastasis. BMDs and behavioral signs were measured prior to each daily drug/vehicle administration to avoid acute effects or interactions with the anesthetic cocktail. Although chronic pamidronate treatment was the last intervention tested in this study, it is presented first in the results for clarity.
4
Osteogenic Differentiation Assay Reagents
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The following were purchased from ThermoFisher Scientific (Carlsbad, CA, USA): MEMα with or without phenol red; MEMα with or without ascorbic acid (vitamin C); MEMα with or without calcium (Ca2+); penicillin/streptomycin; L-glutamine; TaqMan gene expression assays for ALPG (Hs00741068_g1), ALPI (Hs00357579_g1), ALPL (Hs10129144_m1), ALPP (Hs00740632_gH), GAPDH (Hs99999905_m1); universal PCR master mix. Recombinant human ALPL was purchased from R&D Systems (Minneapolis, MN, USA). Rabbit anti-human ALPL antibody, calf intestinal ALP (CIP, ≥10 DEA U/mg), disodium β-glycerophosphate (βGP), phospho(enol)pyruvate monosodium (PEP), pamidronate, dexamethasone, and Alizarin Red S solution were purchased from Sigma-Aldrich (St. Louis, MO, USA). CIP (≥10,000 U/mL), shrimp hepatopancreas ALP (≥1000 unit/mL), and p-nitrophenyl phosphate kits were purchased from New England BioLabs (Ipswich, MA, USA). FBS was purchased from Atlanta Biologicals (Flowery Branch, GA, USA). Disodium α-glycerophosphate hydrate (1 M of αGP; Glycophos) was purchased from Fresenius Kabi (Lake Zurich, IL, USA), and glycerophosphoric acid (NSC 9231) was obtained from the Developmental Therapeutics Program (NCI, USA). Collagen Type I, rat tail, stock solution was obtained from BD Biosciences Discovery Labware (Waltham, MA, USA).
5
Feline OSCC and Fibrosarcoma Cell Lines
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A feline OSCC cell line (SCCF1, provided by Dr. Thomas J. Rosol, The Ohio State University) was grown in Williams E media supplemented with 2 mM l -glutamine, 0.05 mg/mL gentamicin, 10 ng/mL epidermal growth factor, 0.01 nM cholera toxin, and 10% fetal bovine serum (FBS). A feline fibrosarcoma (FSA) cell line (FC83, purchased from ATCC) was grown in DMEM supplemented with 10% FBS. Cell cultures were maintained in subconfluent monolayers at 37°C in a 5% CO2 humidified chamber and passaged twice weekly. Pamidronate was purchased from Sigma-Aldrich (St. Louis, MO) and stock solutions (1 mg/mL) were prepared in sterile phosphate-buffered saline (PBS), aliquoted, and frozen at −20°C until use. Cells were cultured in media as described above for controls; no vehicle control was necessary given use of PBS as Pamidronate drug vehicle.
6
Human pol β purification and assay
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Human pol β was expressed and purified44 (link). Chlodronate, etidronate, imidodiphosphate, pamidronate, and pyrophosphate were from Sigma-Aldrich. The β,γ-imido modified nucleoside triphosphate analog, 2′-deoxyguanosine-5′-[(β,γ)-imido]triphosphate (dGMPPNP), was from Jena Bioscience. Chain terminating nucleoside triphosphates; ddCTP was from GE Healthcare, 3′-azido-2′,3′-dideoxythymidine triphosphate (AZTTP) and arabinofuranosylcytosine triphosphate (araCTP) were from Trilink Biotechnologies, and gemcitabine (dFdCTP) was obtained from Jena Bioscience. [α-35S]dATP, [α-32P]dCTP, and [32P]PPi were from Perkin Elmer. Polyethyleneimine (PEI) cellulose thin layer chromatography (TLC) plates containing a fluorescent indicator were purchased from EMD Millipore.
7
Bisphosphonate Preparation and Use
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Zoledronate used in in vitro experiments was kindly provided by Novartis Pharma (Basel, Switzerland). Clodronate, Pamidronate, Ibandronate were purchased from Sigma Aldrich (St. Louis, MO, USA), as well as Zoledronate used in in vivo studies. The structure of bisphosphonates is depicted in Figure 1 . Bisphosphonate stock solutions were prepared in distilled water, adjusted to pH 7.4 and filter-sterilized prior to use. Empty liposomes and Clodrolip were prepared as described before [43] (link). Other reagents were purchased from Sigma Aldrich. All solutions were prepared in distilled water and filter-sterilized prior to use.
8
Synthesis of NIR Fluorescent Gold Nanocluster-Pamidronate Probe
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A gold nanocluster-based Pamidronate NIR fluorescent probe was synthesized according to the methods in our other studies [37 (link),38 ,39 ]. Briefly, fluorescent gold nanoclusters were synthesized using nanoparticle-etching methods. Pamidronate was attached to the carboxylated surface of a nanocluster using an N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC) (Sigma-Aldrich, St. Louis, MO, USA) crosslinker. Briefly, nano-Au-Pam (em 674 nm) was synthesized by immobilizing Pamidronate (Sigma 2371) onto nano-Au particles using a zero-length crosslinking agent, 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC). To link the Pamidronate to nano-Au particles, equal volumes of Au nanoclusters (40 mM), Pamidronate (3 mM), and EDC (80 mM) were mixed at room temperature for 2 h for the crosslinking reaction. To concentrate the conjugated nano-Au-Pam, the reaction solution was loaded in a 30-kDa molecular sieve (Amicon, Millipore, Bedford, MA, USA) and centrifuged to concentrate the conjugated nano-Au-Pam at 3000 rpm for 10 min. The solution was centrifuged twice and washed with phosphate-buffered saline (PBS) (Sigma-Aldrich, MO, USA) to remove any unbound reactants.
9
Osteogenic Differentiation Protocol
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The following were purchased from ThermoFisher Scientific (Carlsbad, CA, USA): MEMα with or without phenol red; MEMα with or without ascorbic acid (vitamin C); MEMα with or without calcium (Ca 2+ ); penicillin/streptomycin; L-glutamine; TaqMan gene expression assays for ALPG (Hs00741068_g1), ALPI (Hs00357579_g1), ALPL (Hs10129144_m1), ALPP (Hs00740632_gH), GAPDH (Hs99999905_m1); universal PCR master mix. Recombinant human ALPL was purchased from R&D Systems (Minneapolis, MN, USA). Rabbit anti-human ALPL antibody, calf intestinal ALP (CIP, ≥10 DEA U/mg), disodium β-glycerophosphate (βGP), phospho(enol)pyruvate monosodium (PEP), pamidronate, dexamethasone, and Alizarin Red S solution were purchased from Sigma-Aldrich (St. Louis, MO, USA). CIP (≥10,000 U/mL), shrimp hepatopancreas ALP (≥1000 unit/mL), and p-nitrophenyl phosphate kits were purchased from New England BioLabs (Ipswich, MA, USA). FBS was purchased from Atlanta Biologicals (Flowery Branch, GA, USA). Disodium α-glycerophosphate hydrate (1 M of αGP; Glycophos) was purchased from Fresenius Kabi (Lake Zurich, IL, USA), and glycerophosphoric acid (NSC 9231) was obtained from the Developmental Therapeutics Program (NCI, USA). Collagen Type I, rat tail, stock solution was obtained from BD Biosciences Discovery Labware (Waltham, MA, USA).
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