An In Vitro Assay to Quantify the Intracellular Growth of Parasite in Macrophages

Published: February 29, 2024

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…

Divulgaciones

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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An In Vitro Assay to Quantify the Intracellular Growth of Parasite in Macrophages. J. Vis. Exp. (Pending Publication), e21974, doi: (2024).

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