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In This Article

  • Overview
  • Protocol
  • Disclosures
  • Materials

Overview

This video showcases a flow cytometric gating strategy utilizing a multi-color panel to analyze cell viability and specific fluorescent-conjugated anti-human antibody markers. This approach makes it possible to determine the percentage of Mycobacterium tuberculosis-infected cell populations accurately.

Protocol

1. Flow cytometry staining of Mtb-infected monocyte-derived cells

NOTE: The following steps must be performed in a BSL-3 facility. The flow cytometry staining could be performed in a 96-well plate instead of tubes.

  1. Detach the Mycobacterium tuberculosis, Mtb-infected cells (and uninfected controls) from the wells in the 6 well plate(s) by incubation with 1 mL of FACS buffer per well for at least 30 min at 37 °C and 5% CO2.
  2. Gently pipette up and down a few times to ensure that the cells are detached. If possible, confirm cell detachment with microscopy. Transfer ....

Disclosures

No conflicts of interest declared.

Materials

NameCompanyCatalog NumberComments
FormaldehydeSigma-AldrichF8775
Goat anti-mouse IgG Alexa Fluor 594 secondary antibodyInvitrogenR37121Secondary antibody for CD64
Goat anti-Rabbit IgG Alexa Fluor 594 secondary antibodyA-11037Secondary antibody for CD163
Fetal bovine serum (FBS)Sigma-AldrichF7524
EDTA (0.5 M)Karolinska University hospital, HuddingeN/A
BD Comp bead plusBD560497

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