Assessment of Leishmania Virulence within Cultured Macrophages

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)

1. 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 keeping them soaked in 70% ethanol. Spray gloves with 70% ethanol to allow sterile handling throughout the bone marrow cell isolation process.
2. Secure the sacrificed mouse ....

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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