Our research provides a useful guide for studying the biochemical interaction between microbacterium tuberculosis and microbial sensors expressing human microphages and will be relevant to the stiffer molecules that play a role in the entry of microbacterium tuberculosis to fibrocytes. Receptor interaction have been studied over the years using approaches that usually employ the use of reporter machines, hermetic molecules, leveled recombinant proteins, knockout, knockdown, lower expression models. These techniques can be challenging, time demanding, and sometimes expensive.
Using the published protocol, we show, for the first time that in macrophages, SLMF1 receptor interacts with mycobacterium tuberculosis, promoting bacterial uptake and leading to endolysosomal maturation. We have overcome difficulties associated with gene expression experiments and we have described a new target for new therapeutic strategies. Our protocol offers two alternatives to detect biochemical interaction between microbacterium tuberculosis and SLMF1 microbial sensor using flow cytometry and fluorescence microscopy, two usually available and fruitfully used techniques.
This methodology has many potential uses and can be easily adapted in other research contexts. We provide a simple protocol that evaluates mycobacterium tuberculosis receptor interaction using whole cell inactivated and sonicated bacteria, which can be performed under BSL2 condition, but easily adapted to other receptors and live strains. If needed, these assays could also be performed under BSL3 conditions with different live pathogens.
To begin, carefully transfer the blood collected from healthy donors to 50 milliliter tubes and dilute it to half its volume with saline. Prepare a density gradient medium and centrifuge the sample to separate peripheral blood mononuclear cells, PBMCs. Collect the PBMCs from the whitish halo.
After counting the cells in a cell counting chamber, perform magnetic selection with CD14 beads to collect the CD14 positive monocytes. Then resuspend the isolated cells in the RPMI 1640 medium. Seed 500 microliters of CD14 positive monocytes at a concentration of one times 10 to the power of six cells per milliliter into each well of a 24 well cell culture plate in serum absence to promote adherence.
After two hours, wash the wells with pre-warmed RPMI 1640 medium to remove non-adherent cells. Differentiate the monocytes into macrophages using one milliliter of complete RPMI 1640 medium for 16 to 18 hours. The next day, wash the wells with one milliliter of PBS and add one milliliter of complete RPMI 1640 media to each well.
Stimulate the macrophages with 10 microliters of sonicated mycobacterium tuberculosis antigens for 24 hours. The following day, using a P-1000 pipette, discard the complete RPMI medium from the wells of the plate. Add cold PBS and pipette up and down to harvest the macrophages.
Transfer the cells to 1.5 milliliter micro centrifuge tubes. Centrifuge the tubes at 500G for five minutes at four degrees Celsius. Discard the supernatant and resuspend the pellet in a supplemented radioimmunoprecipitation assay buffer.
Incubate the suspension on ice for one hour, vortexing every 10 minutes. Centrifuge the suspension at 14, 000G for 15 minutes and collect the supernatant. Incubate one times 10 to the power of six whole inactivated mycobacterium tuberculosis cells with 50 microliters of protein extract from one times 10 to the power of six macrophages overnight at four degrees Celsius in a rotating micro tube holder.
The next day, to stain the protein bacteria complex, add the anti-human SLAMF1 antibody to the micro tube, vortex the mixture, and incubate overnight for 30 minutes at four degrees Celsius in the dark. After washing the protein bacteria complexes with FACS, resuspend them in FACS buffer and acquire the sample on a flow cytometer. Using round nosed surgical tweezers, place 12 millimeter round cover slips into the wells of a 24 well culture plate.
Incubate the cover slip with mycobacterium tuberculosis rhodamine antigens for one hour at 37 degrees Celsius. After incubation, add 100 microliters of protein extract diluted in 300 microliters of PBS and incubate for two hours at room temperature with agitation. Wrap the lid of the culture plate with paraffin film.
Add a 60 microliter drop of previously titrated anti SLAMF1 primary antibody onto the paraffin film covered lid. Carefully remove the cover slip from the plate using curved fine tip surgical tweezers and needles. Following incubation with primary and secondary antibody solution, remove excess liquid and mount the cover slip on a drop of mounting liquid placed on a glass slide.
Place the slide under a fluorescence microscope and observe the cells using appropriate filters. Monocytes were successfully isolated from PBMCs with a purity of 95%or greater, and monocyte derived macrophages were generated following adherence and overnight culture. SLAMF1 surface expression was induced in macrophages stimulated with mycobacterium tuberculosis antigens, and its levels were confirmed using flow cytometry.
The interaction between SLAMF1 and mycobacterium tuberculosis whole cells was demonstrated using a cross-linking assay followed by flow cytometry. The interaction between the SLAMF1 and Mycobacterium tuberculosis antigens was visualized using fluorescence microscopy with co-localization confirmed by merged images.
