Multicolor Flow Cytometry of Malaria Parasites

P. falciparum IEs were DNA-labeled with ethidium bromide and surface-labeled with rat anti-PfEMP1 antibody (anti-HB3VAR21-DBLβ_D4 or anti-HB3VAR34-DBLβ_D4 antibodies; 1:20) and FITC-conjugated secondary rabbit anti-rat IgG (1:150; Vector Labs) as described previously (Joergensen et al., 2010 (link)). Fluorescence data from ethidium bromide–positive cells were collected on an FC500 MPL flow cytometer (Beckman Coulter) and analyzed using WinList, version 9.0 (Verity Software House). hCMEC/D3 and human brain microvascular pericytes were surface-labeled for CD31 and NG2, respectively. Human astrocytes were permeabilized with 0.1% Triton X-100 before staining with glial fibrillary acidic protein. Fluorescence data were collected on a CytoFlex S flow cytometer (Beckman Coulter) and analyzed using FlowJo (Becton Dickinson).

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Adams Y., Olsen R.W., Bengtsson A., Dalgaard N., Zdioruk M., Satpathi S., Behera P.K., Sahu P.K., Lawler S.E., Qvortrup K., Wassmer S.C, & Jensen A.T. (2021). Plasmodium falciparum erythrocyte membrane protein 1 variants induce cell swelling and disrupt the blood–brain barrier in cerebral malaria. The Journal of Experimental Medicine, 218(3), e20201266.

Publication 2021

FITC-conjugated secondary rabbit anti-rat IgG WinList, version 9.0 CytoFlex S

Anti antibodies Anti antibody Anti igg Astrocytes Brain Cells Ethidium bromide Fitc Fluorescence Glial fibrillary acidic protein Human Pericytes Rabbit Triton x 100 Vector

Corresponding Organization : University of Copenhagen

Other organizations : Brigham and Women's Hospital, Harvard University, Ispat General Hospital, London School of Hygiene & Tropical Medicine

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Variable analysis

independent variables

Labeling of P. falciparum IEs with ethidium bromide
Surface labeling of P. falciparum IEs with rat anti-PfEMP1 antibody (anti-HB3VAR21-DBLβ_D4 or anti-HB3VAR34-DBLβ_D4 antibodies)

dependent variables

Fluorescence data from ethidium bromide–positive cells
Surface labeling of hCMEC/D3 cells with CD31 antibody
Surface labeling of human brain microvascular pericytes with NG2 antibody
Intracellular labeling of human astrocytes with glial fibrillary acidic protein

control variables

Concentration of secondary rabbit anti-rat IgG antibody (1:150)
Permeabilization of human astrocytes with 0.1% Triton X-100 before staining

controls

Positive control: Surface labeling of P. falciparum IEs with rat anti-PfEMP1 antibody
Negative control: Not explicitly mentioned

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