Peripheral blood specimens were collected from 10 newly diagnosed female BC patients from the Tumor Hospital of Harbin Medical University and from 10 healthy female volunteers from Harbin Medical University. Leukocytes were freshly isolated from peripheral blood using a Human Peripheral Blood Leukocyte Isolation Kit (Solarbio, Beijing, China) within two hours after blood collection. Isolated leukocytes were suspended in 1 mL PBS for the following cell fractionations. B cells were isolated from leukocytes first using Dynabeads® CD19 pan B (Invitrogen, Carlsbad, CA, USA). Then, monocytes were isolated from B cell-depleted leukocytes using Dynabeads® CD14 (Invitrogen, Carlsbad, CA, USA). Finally, T cells were purified from B-cell/monocyte-depleted leukocytes using Dynabeads® CD3. DNA extraction and bisulfite conversion from B cells, T cells, and monocytes were performed immediately after purification and the experimental procedures have been described above.
Dynabeads cd14
Manufactured by Thermo Fisher Scientific
Sourced in United States
Dynabeads® CD14 are magnetic beads coated with antibodies specific to the CD14 surface marker. They are used for the isolation and enrichment of CD14-positive cells from various sample types, such as peripheral blood mononuclear cells (PBMCs).
Automatically generated - may contain errors
Lab products found in correlation
6 protocols using dynabeads cd14
1
Leukocyte Fractionation and DNA Extraction from Breast Cancer
2
Isolation and Stimulation of Monocytes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The use of blood from healthy male non-smoking volunteers was approved by the local ethics committee of the University of Giessen (No. 81/13). Peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation using Leucosep gradients (Greiner Bio-One, Frickenhausen, Germany). CD14+ monocytes were enriched by positive selection using Dynabeads® CD14 (Invitrogen, Karlsruhe, Germany) according to the instructions of the suppliers. Cell preparations were evaluated by flow cytometry (FACSCaliburTM, Becton Dickinson, San Jose, CA, USA) using FITC-labeled monoclonal antibody M5E2 to CD14 (BioLegend, San Diego, CA, USA). Monocyte purity was above 75%. Cells were cultured for 3 h and stimulated with BzATP as described previously (15 (link)) and AAT-P (1 mg/ml) was applied together with BzATP.
3
Monocyte Isolation and Gene Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Circulating CD14-positive monocytes were isolated from fresh blood samples by automated magnetic separation using King Fisher (Thermo) with magnetic beads coated with an anti-CD14 monoclonal antibody (Dynabeads CD14, Invitrogen). Total RNA was extracted using the TRIzol reagent and was purified using a NucleoSpin RNA II kit (Macherey-Nagel) according to the manufacturer's instructions.
Reverse transcription of the RNA and real-time quantitative polymerase chain reaction were performed as previously described. 22 (link) The amount of mRNA for the genes of interest was normalized to those of
Reverse transcription of the RNA and real-time quantitative polymerase chain reaction were performed as previously described. 22 (link) The amount of mRNA for the genes of interest was normalized to those of
4
Differentiation of Human Monocytes to Macrophages
5
Oral Glucose Tolerance Test and PBMC Isolation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Seventy-five grams of glucose was administered orally and blood samples for routine analysis were taken at the time points 0, 30, 60, 90, and 120 min. PBMC were isolated from 9 ml of full blood in time points 0 and 120 min and immunoseparated into CD14 positive and negative subpopulations using Dynabeads CD14 (Thermo Fisher, MA USA) as described before (14 (link)).
6
Endotoxin Response Profiling in Healthy Individuals
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The GENE Study recruited healthy individuals (N = 294, 52% Female, 65% EA, 35% AA) ancestry to a University of Pennsylvania (UPenn) inpatient Clinical and Translational Research Center (CTRC) protocol as previously described (clinicaltrials.gov NCT00953667).[30 (link)] Participants completed an endotoxin challenge (1ng/kg E coli-derived LPS; U.S. standard reference, lot No. CCRE-LOT-1+2, Clinical Center, Pharmacy Department at the National Institutes of Health, Bethesda MD).[30 (link), 31 (link)] Multiple clinical variables were assessed during the visit. Individuals were ranked by their peak clinical inflammatory response (Δ from baseline for fever, plasma TNFα, plasma IL-6). Individuals falling within the top and bottom 5% of responses were designated as “high” or “low” responders respectively. CD14+ monocytes were isolated from whole blood using magnetic bead selection (Dynabeads® CD14, ThermoFisher, Waltham MA), and frozen in TRIzol reagent (ThermoFisher, Waltham MA) for subsequent RNA extraction via standard protocol.[32 ]. The GENE study was approved by UPenn’s Institutional Review Board (IRB), with regulatory oversight by the FDA (LPS: IND# 5984) and an NIH-appointed data-safety and monitoring board. All subjects provided written informed consent.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!