Fluorescence-Based Assay for Investigating Macrophage Receptor Interactions with Bacterial Antigens

Samenvatting

Source: Barbero, A. M., et.al. Fluorescence Assays for the Study of Mycobacterium tuberculosis Interaction with the Immune Receptor SLAMF1. J. Vis. Exp. ( (2025)

This video demonstrates a fluorescence-based assay to study the interaction between a macrophage receptor and Mycobacterium tuberculosis antigens. Fluorescently labeled antigens on a coverslip are incubated with macrophage protein extracts, cross-linked, antibody-labeled, and visualized by overlapping fluorescence signals.

Protocol

1. Mycobacterium tuberculosis -SLAMF1 interaction by fluorescence microscopy

1. Bacteria-protein cross-linking
   1. Introduce 12 mm round coverslips into 24-well culture plates (1 coverslip per well) using round-nosed surgical tweezers.
      NOTE: Clean the coverslips thoroughly with 70% ethanol. If desired, they can be autoclaved or sterilized under UV light for 30 min.
   2. Incubate the coverslip with 400 µL of 10 µg/mL Poly-D-Lysine overnight at 4 °C. Cover the plate with aluminum foil to protect it from light.
   3. The next day, remove the plate from the refrigerator and wash the coverslip twice with 1 mL of water.
   4. Let the coverslip dry.
   5. Dilute 20 µL (2 x 10⁶) of Mtb-R Ags in 180 µL of 1x PBS. The final 200 µL is enough to completely cover a coverslip.
   6. Incubate the coverslip with Mtb-R Ags for 1 h at 37 °C in a cell incubator.
   7. Discard the remaining liquid and wash twice with 1mL of 1x PBS (Phosphate-buffered saline).
      NOTE: In this step, the correct adhesion of Mtb-R to the coverslip can be checked with a fluorescence microscope using the filters corresponding to the red channel.
   8. Add 400 µL of blocking buffer (10% fetal bovine serum (FBS) in 1x Phosphate-buffered saline (PBS) for 30 min at RT in agitation (shaker).
   9. Wash twice with 400 µL of blocking buffer.
   10. Incubate with 100 µL of protein extract diluted in 300 µL of 1x PBS for 2 h at RT in agitation (shaker).
   11. Add 400 µL of 1% formaldehyde diluted in 1x PBS. Incubate for 15 min under agitation (shaker) at RT.
   12. Add 500 µL of glycine 0.125 M diluted in water. Incubate for 5 min under agitation (shaker) at RT.
   13. Wash twice with 1 mL of 1x PBS.
   14. Add 400 µL of 2 mM Ethylene glycol bis(succinimidylsuccinate) (EGS for 1 h at RT in agitation (shaker).
   15. Repeat step 1.1.13
       NOTE: The preparation of the reagents at the time of use is highly recommended.
2. SLAMF1 staining
   1. Wrap the lid of the culture plate in paraffin film.
   2. Add a drop of 60 µL of previously titrated anti-SLAMF1 primary antibody (1:75 dilution) on the paraffin film-covered lid.
   3. Carefully remove the coverslip from the plate using curved fine-tip surgical tweezers and needles.
      NOTE: To facilitate coverslip removal, the needle can be bent at an approximate 45° angle.
   4. Invert the coverslip over the drop containing the antibody. Incubate for 30 min at RT in the dark.
   5. Lift the coverslip and wash twice with the blocking buffer.
   6. Invert the coverslip over a new drop containing the secondary antibody (1:200 dilution). Incubate for 30 min at RT in the dark.
   7. Repeat step 1.2.5.
   8. Remove the excess liquid and mount the coverslip on a drop of mounting liquid placed on a glass slide.
   9. Observe under the fluorescence microscope using adequate filters.
      NOTE: Working under sterile conditions is recommended. Dilute the antibodies in the blocking buffer. Handle the coverslip carefully to avoid scratches that may hinder observation under the microscope. A mounting fluid suitable for fluorescence is highly recommended.

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
Alexa Fluor 488 secondary antibody	Invitrogen	A21121	For fluorescence microscopy
Anti-SLAMF1 primary antibody	BioLegend	306302	For fluorescence microscopy
Aqua-Poly/Mount	Polysciences	18606-20	Mounting media
Coverslips 12mm	HDA	-	For interaction assay by microscopy
EGS	ThermoFisher Scientific	21565	For crosslinking treatment
Formaldehyde	Merck	K47740803613	For crosslinking treatment
Glass slides	Glass Klass	-	For interaction assay by microscopy
Glycine	Sigma	G8898	For crosslinking treatment
Imager.A2	Carl Zeiss	430005-9901-000	Fluorescence microscope with Colibri 7 illumination module
Poly-D-Lysine	Sigma Aldrich	A-003-M	For coverslips treatment
Rhodamine B	Sigma Aldrich	21955	For M. tuberculosis staining