So this method can help to answer key questions in the macrophage biology field, such as how to characterize the state of activation under various environment at the protein level. The main advantage of this technique is that it provides the opportunity to obtain differing expression of many proteins in human macrophages using label-free quantification method. Demonstrating the procedure will be Marie Malier and Magali Court from my laboratory.
To begin, add 15 milliliters of density gradient cell separation solution into each of two 50 milliliter centrifugation tubes so that the solution can warm to room temperature before receiving the LRSC. This is essential as density depends on temperature. Empty the LRSC into a 50 milliliter centrifugation tube.
Add 1x PBS up to 50 milliliters and mix. Then, very slowly, add 25 milliliters of the mix on top of 15 milliliters of equilibrated density gradient solution. Be careful not to mix the phases during this step.
The blood must be added on the density gradient solution without any disturbance of this phase. Centrifuge both centrifugation tubes for 25 minutes at 700 G without breaks. At the end of density gradient centrifugation, the layers from the bottom to top are the erythrocytes and granulocytes, forming the pellet, the density gradient solution phase, a layer of PBMCs, and the diluted plasma.
With a pipette, pass through the plasma phase without aspirating it. Then, collect the PBMC layer into a new 50 milliliter centrifugation tube. Add up to 50 milliliters of 1x PBS to the PBMCs as a washing setup and centrifuge for 10 minutes at 300 G.Aspirate the supernatant and re-suspend the pellet in 40 milliliters of macrophage medium.
After couching the PBMCs in a Malassez chamber, transfer the amount of PBMCs necessary for conducting the experiment to a centrifugation tube. Centrifuge the cells for 10 minutes at 300 G.Aspirate the supernatant and re-suspend the pellet in 80 microliters of the sorting buffer per 10 million PBMCs. Then, add 20 microliters of CD14 microbeads per 10 million PBMCs.
Mix well, and incubate for 15 minutes at 4 degrees Celsius under constant agitation. Following incubation, add one milliliter of sorting buffer per 10 million PBMCs as a washing step, and centrifuge as before. Then, aspirate the supernatant and re-suspend the pellet in 500 microliters of sorting buffer per 100 million PBMCs.
Now, place a column in the magnetic field of the separator. Prepare the column by rinsing it with three milliliters of sorting buffer. Apply the cell suspension onto the column.
Then collect the flow through containing unlabeled cells for subsequent staining, if necessary. Wash the column three times with three milliliters of sorting buffer. Be careful not to dry your column.
Then, place a collection tube under the column and remove it from the separator. Following the wash, pipette five milliliters of sorting buffer into the column. Immediately flush out the magnetically-labeled cells by firmly pushing the plunger into the column.
Finally, repeat these steps with a new column before plating the monocytes and performing the macrophage polarization as described in the text protocol. Maintain the monocytes and the macrophages in an oxygen-controlled environment to perform hypoxic condition analysis. Use a hypoxia working station in order to maintain cells under the desired oxygen partial pressure during the experiment.
When working under low oxygen pressure, it is important to prepare all media and washing buffers under the station, and wait sufficiently to obtain the correct partial pressure in the liquid. Perform cell lysis in a buffer under an adapted hood. Load the protein equivalent of 300, 000 cells for each sample on 4-12%bis-tris acrylamide gels.
Control the duration of the electrophoretic migration to allow each protein sample to split into six gel bands as shown here. After fixing and staining as described in the text protocol, excise the protein bands with a clean scalpel. Dice each excised band before placing them into 500 microliter tubes.
Now, wash the gel slices three times in 200 microliters of 25 millimolar ammonium bicarbonate for 20 minutes at 37 degrees Celsius, discarding the ammonium bicarbonate between each step. Follow this with one wash in 25 millimolar ammonium bicarbonate and acetonitrile. Then, dehydrate the gel pieces with 200 microliters of 100%acetonitrile for 10 minutes.
Incubate each gel piece with 10 millimolar DTT and 25 millimolar ammonium bicarbonate for 45 minutes at 56 degrees Celsius. Then, discard the DTT and incubate the gel piece with 55 millimolar iodoacetamide in 25 millimolar ammonium bicarbonate for 35 minutes in the dark at room temperature. To stop alkylation, discard the used solution and incubate each gel piece with 200 microliters of 10 millimolar DTT in 25 millimolar ammonium bicarbonate for 10 minutes at room temperature.
Wash the gel pieces in 200 microliters of 25 millimolar ammonium bicarbonate. Then, dehydrate with 200 microliters of 100%acetonitrile for 10 minutes. Digest the proteins overnight at 37 degrees Celsius with Trypsin/Lys-C Mix according the manufacturer's instructions.
Extract the resulting peptides from gel pieces by adding 50 microliters of 50%acetonitrile for 15 minutes in low-absorption tubes. After adding 50 microliters of 5%formic acid for 15 minutes, add 50 microliters of 100%acetonitrile for 15 minutes. Pull and dry each fraction in low-absorption tubes to limit absorption of peptides and sample loss.
Store the samples at 80 degrees Celsius until further analysis as detailed in the text protocol. Shown here is the representative purity assessed by flow cytometry following magnetic bead selection of CD14 positive monocytes. Phase contrast imaging of differentiated human macrophages show heterogeneity of obtained morphologies for two different polarizations after nine days of culture.
An example of an off-gel digestion using silver-stained SDS page gels is shown. Evaluation of protein from cell lysis and after in-solution digestion shows the absence of degradation during lysis and the efficiency of the digestion. Shown here is an example of a collision-induced dissociation spectrum of a peptide found at mass-to-charge ratio of 597.29 on the MS spectrum with an electric charge of positive two.
From this spectrum, the corresponding sequence was determined. The final result after analysis is a heat map representing the hierarchical clustering of all polarization states using differentially-expressed proteins. Analysis reveals a cluster of proteins over-expressed in all polarizations in the 3%oxygen condition.
While attempting this procedure, it is important to remember that you have to decide first what kind of fractionation you will perform on new samples to adapt your protocol accordingly. The proteomic approach using this work is complementary to genomic approaches that have been used during the last years in the field of the macrophage biology polarization studies. After its development, this technique paves the way for researcher in the field of immunology to explore the various states of activation of immune cells in humans and also in other mammals.
Don't forget that working with DTT could be hazardous so you need to work under an adapted hood to do this procedure.
