To generate bone marrow derived macrophages for polarization analysis. Femur and tibia bones are isolated from mice and the bone marrow cells are collected. The cells are then cultured in medium containing growth factors to induce macrophage formation.
After seven days of culture, the bone marrow derived macrophages are treated with various stimuli and polarized macrophage. Activation responses are analyzed by flow cytometry real-time PCR and western blos analysis results are obtained that show a high purity of bone marrow derived macrophages with polarized activation responses to either M1 or M two. Stimuli lie.
The implication of this technique is stand toward therapy or diagnosis of diversity associated disease, including insulin resistant and the CROs because T infiltrating macrophage are major player in this context, visual demonstration of this method is critical as acceleration of bone marrow cell is somewhat difficult and the yield is critical for obtaining sufficient studying cell preparation for inducing macrophage formation Working in a sterile hood. Begin this protocol with femur and tibia bones isolated from six to eight week old mice in a tissue culture dish. Rinse the bones in PBS then use scissors to cut open both ends of the bones, taking care not to remove too much while holding the bone with forceps.
Insert a 21 on 23 gauge needle with a 10 milliliter syringe containing cold PBS with 2%heating activated FBS into one end of the bone. Press the plunger to flush the marrow out into a new tissue culture dish. Next, pass the marrow through the same needle four to six times to dissociate the cells.
Then pour the cells into a 70 micron cell strainer to remove cell clumps, bone, hair, and other cells or tissues. Collect the flow through in a 50 milliliter conical tube. Once the solution has passed through the filter, add three volumes of 0.8%ammonium chloride to the flow through and incubate on ice for 10 minutes.
To remove red blood cells following the incubation, spin down the cells at 500 times G for five minutes at four degrees Celsius. After the spin, re suspend the cell pellet in 20 to 50 milliliters of cold PBS with 2%FBS. Count the cells using a hemo cytometer.
Spin down the cells at 500 times G for five minutes of four degrees Celsius. After pouring off the supinate. Proceed to induce BMDM formation as described in the next section of the protocol To induce BMDM formation Resus, suspend the isolated bone marrow cells in BMDM growth medium at a concentration of two times.
Center the six cells per milliliter seed one to two times 10 to the six cells in each well of a six or 12 well tissue culture plate and incubate the cells at 37 degrees Celsius. Seeding at this concentration will facilitate detachment for flow cytometry after three days. Change to fresh BMDM growth medium on day seven.
Formation of mature BMDM is evaluated using flow cytometry analysis and fluorol conjugated antibodies to detect cells expressing CD 11 B and F four 80. After verifying the formation of mature BMDM, change to fresh is cove's modified do echo's medium with 10%FBS and LPS or LPS and interferon gamma for M1 activation or IL four or IL 13 for M two activation. Incubate the cells at 37 degrees Celsius after 24 hours.
Collect the stimulated b MDMs by detaching them from the dish. To do this, remove the medium from the cells and wash with PBS without calcium or magnesium. Then add warm 0.05%trypsin containing 0.48 millimolar, EDTA incubate at 37 degrees Celsius for no more than 10 minutes to avoid loss of surface proteins due to over digestion.
Then wash the cells twice with PBS containing 10%FBS and transfer them to 1.2 milliliter sample tubes. Use antibodies to detect expression of cell surface antigens including CD 11 BF four 80, CD 11 C, CD 2 0 6, CD 69, CD 80, or CD 86 at various time points. Using standard flow cytometry staining procedures using quantitative PCR.
Determine the expression levels of IL one beta TNF alpha and IL six to assess M1 activation or IL 10 IL 13, arginase one and PPAR gamma to assess M two activation. Finally perform western blotting to assess activation of cell signaling pathways involved in activation of M1 or M two macrophages to assess the purity of mature BMDM. Following induction flow cytometric analysis was performed using antibodies against CD 11 BF four 80, CD 11 C and CD 2 0 6 B.MDMs were first gated on FSC and SSC to remove DRI and conjugates.
Mature b MDMs were defined as CD 11 B positive F four 80 positive subpopulations, which are seen in the upper right of this plot and compromised 95 to 99%of the gated population to assess macrophage polarization. Further analysis was performed. First gating was performed to identify tissue infiltrated macrophages, which are CD 11 B positive F four 80 positive cells.
Among those M1 macrophages, which are CD 11 C positive and CD 2 0 6 negative appear in quadrant two, whereas M two macrophages, which are CD 11 C negative and CD 2 0 6 positive appearing quadrant four. To assess macrophage activation cells were incubated with either LPS to induce M1 activation or IL four for M two activation. Upon activation, the size of macrophages increased as evidenced by the shift of FSCA of M1 or M two macrophages at 24 hours after stimulation compared to M zero untreated.
Macrophages activation related surface markers were also analyzed after stimulation. The early responding markers CD 69 was evaluated five or 24 hours post stimulation CD 80 and CD 86 markers were analyzed at 48 hours after stimulation. As shown here, increased abundance of surface CD 69, CD 80, and CD 86 was seen in stimulated macrophages to assess classical and alternative activation of macrophages.
Those stimulated with either LPS or IL four for 24 hours were collected and total RNA was extracted for analysis by quantitative real-time PCR as shown here. Elevated arginase one and PPAR gamma expression were detected in M two macrophages as compared to untreated macrophages expression of pro-inflammatory cytokines. Interleukin one beta TNF alpha increased in M1 macrophages to assess activation of the NF kappa B pathway by LPS antibodies against P 65 and phospho related P 65 were used in western blot analysis as shown here.
A stronger band against phosphorylated P 65 is seen with LPS treatment taken. Together, these data indicate bone marrow derived macrophages with polarized activation responses to either M1 or M two stimuli. After watching this video, you should have a good understanding of how to generate bone marrow derived macrophage, followed by polarized activation analysis.
While attempting this procedure, it's important to remember to evaluate the purity of differentiated macrophage before testing their response. Following this procedure, a method like transient transfection of genes or SHRA can be performed in order to answer additional question like, how are genes involved in regulating macrophage ized activation?
