Source: Sarkar, A. et al., Quantification of Intracellular Growth Inside Macrophages is a Fast and Reliable Method for Assessing the Virulence of Leishmania Parasites. J. Vis. Exp. (2018)
This video investigates Leishmania's virulence by treating macrophages with metacyclic promastigotes, resulting in the formation of intracellular amastigotes. The confirmation of Leishmania's virulence is achieved through nucleic acid fluorescent dye staining. This insight can contribute to the development of new therapies for leishmaniasis.
All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Isolation and Differentiation of Bone Marrow-derived Macrophages (BMMs)
2. Plating BMMs on Coverslips for Infection (DAY 7 or 8)
3. Purification of Infective Forms of Leishmania amazonensis
NOTE: Prepare Leishmania for infections- purify metacyclic promastigotes from stationary promastigote cultures, or differentiate promastigotes in culture into amastigote form using standard L. amazonensis axenic differentiation protocol.
4. Infection with L. amazonensis
5. 4′,6-diamidino-2-phenylindole (DAPI) Staining and Coverslip Mounting
6. Infection Quantification
Figure 1: A representative illustration of BMM infection with virulent axenic amastigotes (A) and non-virulent log-phase promastigotes (B) of L. amazonensis. Immunofluorescence images of macrophages isolated from BALB/c mice, 1 h and 48 h following infection. Infected macrophages were processed for immunofluorescence as described in the protocol. PV membranes were stained with rat anti-mouse Lamp1 monoclonal antibody (1:1,000 dilution) for 1 h, followed by 1 h incubation with anti-rabbit fluorescent IgG (1:500 dilution). Parasite staining was performed by incubating coverslips with mouse polyclonal antibodies generated against axenic L. amazonensis amastigotes, followed by anti-mouse IgG red dye (1:500 dilution) 6. All coverslips were further treated with DAPI for staining nuclei. (A) Formation of distinct PVs harboring multiple amastigotes at 48 h time point is characteristic of a successful Leishmania infection with axenic amastigotes. (B) The absence of distinct PV formation and replicating amastigotes at a 48 h time point typifies a lack of virulence in promastigotes from log-phase culture. Red indicates anti-Leishmania staining, green indicates anti-Lamp1, blue indicates DAPI-stained DNA, and yellow indicates a merge of anti-Lamp1 and DAPI stains. Bars, 5µm. This figure has been modified from Mittra, B. et al., 2013. Originally published in J. Exp. Med.
The authors have nothing to disclose.
6 well cell culture plate | Cellstar | 657160 | |
Adenine | Acros Organics | AC147440250 | |
Aerosol Barrier Pipet Tips (100-1000 μL) | Fisherbrand | 02-707-404 | |
Aerosol Barrier Pipet Tips (20-200 μL) | Fisherbrand | 02-707-430 | |
Aerosol Barrier Pipet Tips (2-20 μL) | Fisherbrand | 02-707-432 | |
Bard-Parker Rib-Back Carbon Steel Surgical Blade #10 | Aspen Surgical | 371110 | |
BD Luer-Lok Tip 10 mL Syringe | Becton Dickinson (BD) | 309604 | |
BD Precisionglide Needle, 25G | Becton Dickinson (BD) | 305124 | |
Cell Culture Dish 35 mm x 10 mm | Cellstar | 627 160 | |
Cell Culture Flask | Cellstar | 660175 | |
Cover Glasses: 12 mm circles | Fisherbrand | 12-545-80 | |
DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride) | Invitrogen | D1306 | |
D-Biotin | J.T. Baker | C272-00 | |
EDTA | Sigma Aldrich | EDS | |
Ethyl alcohol 200 proof | Pharmco-AAPER | 111000200 | |
Falcon 100 mm x 15 mm non-TC-treated polystyrene Petri dish | Corning | 351029 | |
Fetal Bovine Serum | Seradigm | 1500-500 | |
Ficoll400 | Sigma Aldrich | F8016 | |
Fluorescence Microscope | Nikon | E200 | |
Goat anti-mouse IgG Texas red | Invitrogen | T-862 | |
Goat anti-rabbit IgG AlexaFluor488 | Invitrogen | A-11034 | |
Hemin | Tokyo Chemical Industry Co. LTD | H0008 | |
HEPES (1M) | Gibco | 15630-080 | |
Isoton II Diluent | Beckman Coulter | 8546719 | |
L-Glutamine | Gemini | 400-106 | |
Medium 199 (10X) | Gibco | 11825-015 | |
Na pyruvate (100 mM) | Gibco | 11360-070 | |
Paraformaldehyde | Alfa Aesar | 43368 | |
Penicillin/Streptomycin | Gemini | 400-109 | |
Phosphate Buffered Saline (-/-) | ThermoFisher | 14200166 | |
Polypropyline conical Centrifuge Tubes 15 mL | Cellstar | 188 271 | |
Polypropyline conical Centrifuge Tubes 50 mL | Cellstar | 227 261 | |
ProLong Gold antifade reagent | ThermoFisher | P36930 | |
Rat anti-mouse Lamp-1 antibody | Developmental Studies Hybridoma Bank | 1D4B | |
Recombinant Human M-CSF | PeproTech | 300-25 | |
Reichert Bright-Line Hemocytometer | Hausser Scientific | 1492 | |
RPMI Medium 1640 (1X) | Gibco | 11875-093 | |
Triton X-100 Surfactant | Millipore Sigma | TX1568-1 | |
Trypan Blue | Sigma Aldrich | T8154 | |
Delicate Scissors, 4 1/2" | VWR | 82027-582 | |
Dissecting Forceps, Fine Tip | VWR | 82027-386 | |
Microscope Slides | VWR | 16004-368 | |
Z1 Coulter Particle Counter, Dual Threshold | Beckman Coulter | 6605699 |