Assessment of Intracellular Growth of Coxiella Mutants within Eukaryotic Cells

Published: November 30, 2023

Abstract

Source: Martinez, E. et al., Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants. J. Vis. Exp. (2015)

This video demonstrates a method for assessing the intracellular growth of GFP-tagged Coxiella mutants within cultured epithelial cells. The mutant phenotype is visualized using immunostaining and epifluorescence microscopy, allowing the study of the effect of the mutation on intracellular growth.

Protocol

1. Generation of a library of green fluorescence protein, GFP-tagged Coxiella transposon mutants Manipulate Coxiella burnetii RSA439 NMII in a biosafety containment level 2 (BSL-2) in a microbial safety cabinet (MSC) in compliance with local rules. If compatible with the bacterial model used, repeat steps from 1.4.1 to 1.4.4 to increase the probability of obtaining clonal mutants. A typical mutant library is composed (at least) of a number o…

Açıklamalar

The authors have nothing to disclose.

Materials

Citric acid Sigma C0759-500G ACCM-2 medium component
Sodium citrate Sigma S4641-500G ACCM-2 medium component
Potassium phosphate Sigma 60218-100G ACCM-2 medium component
Magnesium chloride Sigma M2670-100G ACCM-2 medium component
Calcium chloride Sigma C5080-500G ACCM-2 medium component
Iron sulfate Sigma F8633-250G ACCM-2 medium component
Sodium chloride Sigma S9625-500G ACCM-2 medium component
L-cysteine Sigma C6852-25G ACCM-2 medium component
Bacto Neopeptone BD (Beckton-Dickinson) 211681 ACCM-2 medium component
Casamino acids BD (Beckton-Dickinson) 223050 ACCM-2 medium component
Methyl-B-Cyclodextrin Sigma C4555-10G ACCM-2 medium component
RPMI w/glutamax Gibco 61870-010 ACCM-2 medium component
RPMI w/glutamax, without phenol red Gibco 32404-014 For cell culture and infection
Fetal Bovine Serum GE healthcare SH30071 For cell culture
Trypsin EDTA (0.25%) Life Technologies 25200-056 For cell culture
Saponin Sigma 47036 For immunofluorescence staining
Ammonium chloride Sigma A9434 For immunofluorescence staining
Bovine Serum Albumine Sigma A2153 For immunofluorescence staining
Electroporation cuvette 0.1 cm Eurogentec ce0001-50 For Coxiella transformation
Trackmates screw top tubes with caps  Thermo Scientific 3741 2D barcoded screwcap
96-well microplate with black walls and bottom  Greiner 655076 Flat dark bottom, for dsDNA quantitation
96-well microplate, clear, black walls Greiner 655090 Flat transparent bottom, for cell culture and imaging
96-well PCR microplate Biorad 2239441 DNAse/RNAse free
96-well plate, Deepwell Labcon 949481 For Coxiella mutants infections
PicoGreen Life Technologies P7581 For dsDNA quantitation
Triton X-100  Sigma T9284-500ML For dsDNA quantitation
Phusion High fidelity DNA Polymerase New England Biolabs M0530L For single primer colony PCR
Celltracker Red CMTPX Life Technologies C34552 For imaging
Anti-LAMP1 antibody Sigma 94403-1ML For immunofluorescence staining
Hoechst 33258 Sigma L1418 For imaging
Fluorescence microplate reader Infinite 200 Pro Tecan
Epifluorescence automated microscope Cellomics Thermo Scientific

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Bu Makaleden Alıntı Yapın
Assessment of Intracellular Growth of Coxiella Mutants within Eukaryotic Cells. J. Vis. Exp. (Pending Publication), e21794, doi: (2023).

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