Csf1r-iCre mice were provided by Dr. Elaine Lin (Deng et al., 2010 (link)) on the FVB background and Stat5fl/fl mice were provided by Dr. Lothar Hennighausen (Cui et al., 2004 (link)). Wild-type FVB mice were purchased from Harlan Laboratories and the Stat5fl/fl mice were backcrossed to the FVB/N background and backcrossing was verified using congenic analysis (IDEXX-RADIL, Columbia, MO). For iFGFR1 activation, mice were injected twice weekly with 1 mg/kg B/B homodimerizer (Clontech) by intraperitoneal injection as previously described (Schwertfeger et al., 2006b (link); Welm et al., 2002 (link)). Daily estrous staging was performed as previously described using crystal violet-stained cytology of vaginal lavage fluid (McLean et al., 2012 (link)). Two hours prior to sacrifice, mice were injected with 30 mg/kg BrdU by intraperitoneal injection. All animal care and procedures were approved by the Institutional Animal Care and Use Committee of the University of Minnesota and were in accordance with the procedures detailed in the Guide for Care and Use of Laboratory Animals.
B b homodimerizer
Manufactured by Takara Bio
Sourced in United States
The B/B homodimerizer is a laboratory reagent designed to facilitate the dimerization of two identical proteins or protein domains. It functions by binding to specific binding domains within the target proteins, inducing their homodimerization. The core function of the B/B homodimerizer is to enable the controlled dimerization of proteins in experimental settings.
Automatically generated - may contain errors
Lab products found in correlation
Ap20187, by Takara Bio (3 mentions)
Phosal 50 pg, by Lipoid (2 mentions)
Mapt p301s ps19 ps19 mice, by Jackson ImmunoResearch (2 mentions)
Peg400, by Merck Group (2 mentions)
Celltiter 96 aqueous one solution cell proliferation assay, by Promega (2 mentions)
Matrigel, by BD (2 mentions)
Hl5c medium, by Formedium (1 mentions)
Opti mem, by Thermo Fisher Scientific (1 mentions)
X tremegene 9 dna transfection reagent, by Roche (1 mentions)
Mts reagent, by Promega (1 mentions)
Bovine thrombin, by Merck Group (1 mentions)
Bay 11 7085, by Cayman Chemical (1 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (1 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions)
Penicillin streptavidin, by Thermo Fisher Scientific (1 mentions)
Dmem medium, by Thermo Fisher Scientific (1 mentions)
Annexin 5 apc, by BioLegend (1 mentions)
Nih3t3 cells, by Leibniz Institute DSMZ (1 mentions)
Annexin 5 fitc, by Immunotools (1 mentions)
Zvad fmk, by Bachem (1 mentions)
34 protocols using b b homodimerizer
1
Conditional Genetic Manipulation of Mice
2
Inducible Dictyostelium Dimerization Assay
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The axenic Dictyostelium discoideum wild-type strain was used for all experiments. Cells were grown in HL5-C medium (Formedium) at 22 °C. The indicated constructs were transformed in AX2 cells by electroporation and selected with either 10 µg/mL geneticin or 50 µg/mL hygromycin B. To induce expression from the tetracycline inducible vectors, the cells were overnight incubated with 1 µg/mL doxycycline. Dimerization of FKBP12-GFP was induced by incubation with B/B homodimerizer (Clontech, also called AP20187) to a final concentration of 1 µM for 3 h.
3
Caspase-9 Induction in CAF-Breast Cancer Spheroids
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10 nM B/B homodimerizer (Clontech) was utilized to induce caspase-9 expression in CAF-hTERT-Casp9-GFP in co-culture experiments. 0.5 mM B/B homodimerizer stock (in ethanol, vehicle) was diluted to 10 nM in spheroid co-culture medium and spheroids were treated with B/B homodimerizer at 0 h or 12 h post collagen polymerization. For 2D pre-education experiments, caspase-9 induction in CAF01-hTERT-Casp9-GFP occurred following 3 days of co-culture. CAF-educated MDA-MB-231 or SUM-159 were isolated at 24 h following the addition of B/B homodimerizer and used to form mono-culture breast cancer spheroids.
4
Induction of Casp4-mediated Pyroptosis in HeLa and HEK 293T Cells
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HeLa (stably expressing Flag–GSDMD–V5 and DmrB–Casp4) and HEK 293T cells were seeded in 96-well plates at a density of 8.5 × 103 and 1.2 × 104 cells per well, respectively, a day before transfection. The cells were then transiently transfected with 50 ng per well of a pLVX DNA plasmid encoding wild-type or different mNINJ1 mutants, using X-tremeGENE 9 DNA Transfection Reagent (Roche) according to the manufacturer’s instruction. To induce the transgene expression, the medium was supplemented with Dox (1 μg ml−1) for 16 h (HeLa) or 48 h (HEK 293T). In HeLa cells, medium was replaced with opti-MEM (Gibco) and cells were incubated with 20 nM B/B homodimerizer (Takara, Clontech) for 1.5 h to activate DmrB–Casp4.
5
Targeting Senescent Cells in Aging Mice
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All animal protocols were approved by the Institutional Animal Care and Use Committee (IACUC), and all experiments were performed in accordance with IACUC guidelines. Mice were housed in ventilated cages in a pathogen-free facility (12-hour light/dark cycle, 23 °C) and had access to food (standard mouse diet, Lab Diet 5053, St. Louis, MO) and water ad libitum. Mouse experiments for a genetic targeting approach of senescent cells have been described by our group earlier37 (link). Briefly, old (20 months) female mice were injected intraperitoneally with vehicle (4% of 100% EtOH, 10%PEG400, 86% of 2% Tween 20 in deionized Water) or AP20187 (B/B homodimerizer, Clontech; 10 mg of AP20187 per kg body mass) twice weekly at the age of 20 months for a total of 4 months (old mice were sacrificed at 24 months of age). In addition, young (6-month) INK-ATTAC mice were used as a control comparison cohort.
6
Cell Proliferation Assay for FGFR1 Activation
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BoM-1833 cells and HC-11/R1 cells were plated at 10,000 cells/well in a 96-well plate. After 24 hours, BGJ398 dissolved in DMSO was added at the indicated concentrations. 30 nM B/B homodimerizer (Clontech) was added to the HC-11/R1 cells to activate FGFR1. Plates were incubated for 72 hours prior to addition of MTS reagent per manufacturer’s instructions (Promega).
7
Caspase-11 Activation and GSDMD Cleavage Assay
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The DmrB-ΔCARD-caspase-11 mutants were cloned into the pEF6 vector. HEK293T cells (ATCC CRL-3216) were transfected with these constructs using lipofectamine, and cells were reseeded at 1 × 106 cells/ml. Transfected cells were then incubated in opti-MEM containing the 500 nM of the B/B Homodimerizer (AP20187; Clontech) for 30 min. The medium was then replaced with caspase activity buffer (200 mM NaCl, 50 mM Hepes pH 8.0, 50 mM KCl, 100 μg/ml digitonin, 10 mM DTT) supplemented with 100 μM AcLEHD-afc or V5-GSDMD-expressing HEK293T cell extract. For experiments in which caspase-11 was cleaved with thrombin, 20 U/ml bovine thrombin (Sigma-Aldrich) was added to the caspase activity buffer 15 min before V5-GSDMD addition, or the same time as AcLEHD-afc addition. Hydrolysis of the caspase-11 substrate AcLEHD-afc was monitored at 37°C at regular time intervals using the M1000 TECAN spectrofluorometer (400 nm excitation, 505 nm emission). V5-GSDMD cleavage was monitored after 2 h or as indicated. Cell extracts and supernatants were precipitated using methanol/chloroform and analysed by immunoblotting using standard procedures (Gross, 2011 ).
8
Senolytic Intervention in Tauopathy Mice
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MAPT P301S PS19 (PS19) mice were purchased from The Jackson Laboratory (stock #008169) and bred to C57BL/6 for three generations. C57BL/6 ATTAC transgenic mice are as described3 (link),4 (link). Male PS19 mice were bred to ATTAC females to generate cohorts of ATTAC and PS19;ATTAC mice. All mice were on a pure C57BL/6 genetic background. Mice from this cohort were randomly assigned to receive AP20187 (AP; B/B homodimerizer; Clontech) or vehicle twice a week beginning at weaning age (3 weeks). Dosing of AP was 2.0 mg kg–1 body weight. 6-month-old short-term AP pulse treated animals (Extended Data Fig. 6a ) received a dose of 10 mg kg–1 body weight for 5 consecutive days prior to tissue collection. Senolytic intervention was performed in C57BL/6 WT and PS19 animals. At weaning, mice were assigned to receive either ABT263 (Cayman, 923564–51-6) or vehicle (Phosal 50 PG, Lipoid NC0130871 – 60%; PEG400, Sigma 91893 – 30%, EtOH – 10%). ABT263 was administered by oral gavage at a dose of 50 mg kg–1 body on a repeating regiment of five consecutive days of treatment followed by 16 days of rest. Animals were housed in a 12h L/D cycle environment in pathogen-free barrier conditions as described in detail3 (link). Compliance with relevant ethical regulations and all animal procedures were reviewed and approved by the Mayo Clinic Institutional Animal Care and Use Committee.
9
Modulating Subcellular Protein Dynamics
10
Assessing Suicide Gene Functionality in iPSCs and Macrophages
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1-2x105 iPSCs were seeded on MEF- or Matrigel-coated 12-well tissue culture plates and grown to sub-confluency before the functionality of the suicide gene was assessed by adding the CID (AP20187, B/B Homodimerizer, Clontech Laboratories, CA, USA) for 24 h with concentrations ranging from 0 nM- 10 nM. Twenty-four hours after supplementation with CID the cells were reseeded onto a 6-well tissue culture plate, kept for 14 days and an appearance of iPSC colonies was observed.
Monocytes/macrophages were terminally differentiated in differentiation medium II for 5 days and seeded in 96-well tissue culture plates at a density of approximately 6-10x104 cells/well before being exposed to 0 nM- 10 nM AP20187 for 24 h. For flow cytometric analysis, cells were dissociated into single cells using TrypLE and stained with PI (according to the manufacturer´s instruction). Additionally, pictures of the cells in culture plates were taken at the indicated time points.
Monocytes/macrophages were terminally differentiated in differentiation medium II for 5 days and seeded in 96-well tissue culture plates at a density of approximately 6-10x104 cells/well before being exposed to 0 nM- 10 nM AP20187 for 24 h. For flow cytometric analysis, cells were dissociated into single cells using TrypLE and stained with PI (according to the manufacturer´s instruction). Additionally, pictures of the cells in culture plates were taken at the indicated time points.
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