Isolation of Murine Peritoneal Macrophages to Carry Out Gene Expression Analysis Upon Toll-like Receptors Stimulation

The overall goal of this procedure is to isolate murn peritoneal macrophages and to perform gene expression analysis upon the stimulation of toll-like receptors. This is accomplished by first injecting. 3.8%of brewer thi glyco medium into the peritoneal cavity of the mouse to increase monocyte migration into the peritoneum and therefore increase macrophage yield after three days, euthanize the mouse and inject cold ECCOs phosphate buffer saline into the peritoneal cavity to harvest the cells.

Following a gentle massage on the peritoneal cavity, the peritoneal fluid is carefully aspirated. Next, the obtained cells are cultured and the elicited peritoneal macrophages are characterized by flow cytometry. The final step is to stimulate these macrophages with different toll-like receptor or TLR ligands and isolate the RNA from the samples.

Ultimately, quantitative reverse transcriptase polymerase chain reaction is used to measure expression of the gene of interest. The goal of the following procedure is to isolate, mirroring peritoneal microphages using brewer's TIO glycol medium. This medium will increase monocyte migration into the peritoneum and therefore increase microphage yield.

Once isolated microphages will be stimulated with different toll-like receptor ligands to study the molecular regulation of hepcidin as our gene of Pinterest. To begin, prepare 3.8%brewer's bio glyco eight medium as described in the text protocol using one milliliter syringes attached to 23 gauge needles. Inject one milliliter of 3.8%brewer's bio glycol eight medium into the peritoneal cavity of each mouse, and wait for three days.

Use a new syringe and needle for each mouse following mouse anesthetization and euthanasia as described in the text protocol, soak the abdomen of each mouse with 70%ethanol. Perform a lateral incision along the bottom midline of the peritoneum with scissors. Use forceps to pull back the abdominal skin and expose the transparent peritoneal skin.

Then inject five milliliters of cold ECCOs phosphate buffered saline into the peritoneal cavity of each mouse with five milliliter syringes attached to 20 gauge needles. Perform a gentle massage on the peritoneal cavity and then aspirate the fluid carefully without puncturing any organ. Remove the needle and dispense the peritoneal fluid into 50 milliliter conical centrifuge tubes.

Next centrifuge the collected peritoneal fluid for 10 minutes at 400 times G or 1500 RPM in a refrigerated centrifuge, cells should be kept cold throughout the procedure. Discard the supernatant and resuspend the cell pellet in RPMI 1640 medium. Using a hemo cytometer, count the cells and adjust the samples to a cell density of 1 million cells per milliliter.

Characterize the phenotype of isolated cells by flow cytometry to confirm macrophage enrichment. Using 1 million cells per mouse and antibodies against F four 80, which is a surface antigen expressed on macrophages directly. After isolation, add 1 million cells into each well of a six.

Well plate. Leave the murine peritoneal macrophages to adhere to the wells by culturing them for one to two hours at 37 degrees Celsius. Following incubation, remove non-adherent cells by gently watching three times with warm phosphate buffered saline.

Then add 900 microliters of serum free DMEM to the cells. Prepare 10 x stock solutions of each toll-like receptor or TLR ligand as listed in the text protocol. Then add 100 microliters of the stock solutions to each well, thus performing a tenfold dilution culture.

The cells for 24 hours in the presence of the TLR ligands. To perform the RNA isolation first, remove all medium and lyce the cells directly in the six well plates. By adding one milliliter of triol reagent to each well and passing the cell lysate several times through a pipette.

Then incubate the homogenized samples for five to 10 minutes at room temperature to permit the complete dissociation of nuclear protein complexes. Following incubation, transfer the lysate into 1.5 milliliter RNAs and DNA spree micro centrifuge tubes, and add 0.2 milliliters of chloroform per one milliliter of triol reagent. Shake the tubes vigorously by hand for 15 seconds before incubating them at room temperature for five to 10 minutes.

Then centrifuge at 12, 000 times G for 15 minutes at four degrees Celsius. Observe the mixture separating into a lower red phenol chloroform phase, an interphase and a colorless upper aqueous phase. RNA remains exclusively in the aqueous phase.

Carefully transfer the upper aqueous phase to a fresh tube without disturbing the interphase phase. Precipitate the RNA from it by mixing with 0.5 milliliters of isopropyl alcohol after incubating the samples at room temperature for 10 minutes. Centrifuge at 12, 000 times G for 10 minutes at four degrees Celsius.

The RNA precipitates forming a white pellet on the side and bottom of the tube. Remove the supernatant and wash the RNA pellet once with one milliliter of 75%ethanol. Mix the samples by vortexing and then centrifuge at 7, 500 times G for five minutes at four degrees Celsius.

After removing the supernatant, briefly air dry the RNA for 10 minutes. Then dissolve the RNA in 100 microliters of RNA free water and incubate for 10 minutes at 55 degrees Celsius. Next, quantify the RNA using a spectrophotometer to do so.

Dilute samples 100 times in RNA free water and read at wavelengths of 260 nanometers equalize RNA concentrations and synthesize CD NA as per manufacturer's instructions. Using the R-T-P-C-R system for first strand CD NA synthesis kit. Then measure gene Mr.NA levels by realtime PCR in a real-time, DNA detection system, normalizing gene expression levels with two housekeeping genes.

The isolated mirroring peritoneal macrophages were characterized by flow cytometry to determine the percentage of isolated macrophage and to distinguish them among cells obtained during the isolation process. The percentage of cells expressing the antigen F four 80 was consistently found to be above 95%The isolated cells were then treated with several TLR ligands and mRNA. Levels of hepcidin were measured by R-T-P-C-R tolike receptor one and two, tolike receptor four.

An olic receptor six and two ligands were capable of stimulating hepcidin mRNA in mirroring macrophages. Together these results demonstrate the usefulness of this protocol to successfully isolate, mirroring peritoneal macrophages and to precisely investigate the molecular regulation of gene expression. After watching this video, you will have a good understanding of how to isolate, identify, and culture mirroring peritoneal macrophages.