An In Vitro Assay to Quantify the Intracellular Growth of Parasite in 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 demonstrates an assay to quantify the intracellular growth of parasites in bone marrow-derived macrophages (BMMs). The macrophage cells are infected with the motile and non-motile invasive stages of Leishmania, metacyclic promastigotes and amastigotes, respectively. The parasites invade the macrophages, getting internalized into a parasitophorous vacuole and multiplying inside. The intracellular parasite growth is quantified using a microscope upon fluorescently staining the macrophage and parasite nuclei.

Protocol

1. Plating BMMs on Coverslips for Infection Prepare 6-well tissue culture plates by placing four (4) sterile 12 mm glass coverslips (autoclaved and stored in a closed glass container) in the bottom of each empty well. NOTE: Pick each sterile 12 mm coverslip up with an aspirating tip fitted to a vacuum line. Place coverslips into the wells without overlap, releasing them at the desired position by pinching the tube to interrupt the vacuum. Aspirate media (and…

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 were stained with r…

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