Assessment of Leishmania Virulence within Cultured Macrophages

Published: November 30, 2023

Abstract

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.

Protocol

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) Day 0: Sacrifice 4 – 6-week-old female C57BL/6 or BALB/c mouse in a CO2 chamber and confirm death via cervical dislocation. Ensure sterilization of scissors and forceps by k…

Representative Results

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…

開示

The authors have nothing to disclose.

Materials

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

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記事を引用
Assessment of Leishmania Virulence within Cultured Macrophages. J. Vis. Exp. (Pending Publication), e21792, doi: (2023).

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