5×105 WBM cells isolated from either ControlVav or Jarid2-KOVav was transplanted into lethally irradiated recipients along with 5×105 wild-type CD45.1 WBM by retro-orbital injection. Eight weeks post-transplant, a single dose of 5-FU (150 mg/kg; Sigma-Aldrich F6627–1G) was injected at the same time as BrdU (3.33mg/mouse; Sigma #B5002) via intra-peritoneal route, and BrdU (0.8mg/mL) was supplemented in the drinking water for 96 hr prior to harvest. For unmanipulated mice, two doses of 5-FU (150 mg/kg) were injected seven days apart, with BrdU (3.33mg/mouse) injected via intra-peritoneal route at the same time as the second 5-FU injection, and BrdU (0.8mg/mL) was supplemented in the drinking water for 96 hr prior to harvest. For BrdU analysis, 10×106 WBM cells were stained with cell surface markers overnight at 4oC followed by staining with anti-BrdU-APC (BD Pharmingen #51–9000019AK) according to the manufacturer’s directions.
Sigma b5002
Manufactured by Merck Group
Sourced in United States
Sigma B5002 is a laboratory product manufactured by Merck Group. It is a specialized instrument designed for scientific research and analysis. The core function of this product is to perform specific tasks within a laboratory setting. However, a detailed description of its intended use cannot be provided while maintaining an unbiased and factual approach.
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Lab products found in correlation
4 protocols using sigma b5002
1
Hematopoietic Stem Cell Regeneration
2
Islet Morphometric Analysis and Metabolic Assays
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Insulin measurements from plasma and pancreatic extracts were measured by radioimmunoassay (RIA) (Millipore), and blood glucose and luciferase assays were measured as described previously (Poy et al., 2009 (link)). Islet morphometric analysis after intraperitoneal injections of BrdU on four consecutive days (50 μg/g BW, Sigma B5002) was performed on 8 μm sections of paraffin-embedded pancreas approximately 150–200 μm apart. Sections were dewaxed, washed, and stained for insulin (Dako A0564), glucagon (Millipore AB932), BrdU (Abcam ab6326), Ki-67 (Dako), or TUNEL (Roche cat. no.12156792910). Cell numbers from all islets in 3–7 sections were counted with ImageJ software from 20X images obtained using a Zeiss LSM700 (Schneider et al., 2012 (link)). β cell mass was measured as the ratio of insulin-positive cell area to the total tissue area, multiplied by the weight of the pancreas using Imaris software (Bitplane). In vivo insulin release and glucose (GTT) or insulin (ITT) tolerance tests were performed following a 6 hr fast and injected intraperitoneally with either glucose (2 g/kg BW) or insulin (0.75 U/kg BW).
3
Dual DNA Labeling in Mouse Embryos
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Pregnant dams (E14.5 or E16.5) were injected intraperitoneally with 120 mg IdU per kg body weight at time (T) 0. At T = 1.5 hours, dams were injected with 100 mg BrdU (Sigma B5002, Sigma-Aldrich, St Louis, MO, USA) per kg body weight. At T = 2 hours, dams were culled and embryos were fixed in cold 4% PFA. A mouse antibody against IdU and BrdU (BD) detected both thymidine analogs, and a rat antibody specific to BrdU (Serotec, Raleigh, NC, USA) detected BrdU alone [50 (link)].
4
BrdU Injections for Cell Proliferation Analysis
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Bromodeoxyuridine (BrdU) injections were used for two different experiments. To evaluate the short-term cell division, the animals (n = 5 per group) received an intraperitoneal injection of BrdU (50 mg/kg; Sigma B5002, St. Louis, MO, USA) dissolved in 0.007 N NaOH solution, 2 h before sacrifice (Xie et al., 2016 (link)). To label the cell proliferation at the long term, the mice (n = 5 per group) received an intraperitoneal injection of 50 mg/kg BrdU every 8 h for the first 3 days (Gonzalez-Perez et al., 2011 (link)). These BrdU injections were done 72 h after vibrissal fulguration to minimize the effect of inflammatory cytokines triggered by the surgical procedure.
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