Assessing Pneumococcal Cytotoxicity in A549 Cells

A549 respiratory epithelial cells (obtained from ATCC, catalog number CCL-185) were grown in minimum essential medium (MEM) with 10% fetal bovine serum as described [20 (link)]. Cell monolayers (80–90% confluent in sterile 24-well plates) were incubated in MEM without serum overnight prior to stimulation with ~10⁸ cfu of the indicated S. pneumoniae strains for 12 hrs. LDH cytotoxicity assay (Roche) was performed as per the manufacturer’s instructions, and values were normalized to 0% (vehicle control) and 100% lysis (1% Triton X-100) controls as previously described [21 (link)].

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Amaral F.E., Parker D., Randis T.M., Kulkarni R., Prince A.S., Shirasu-Hiza M.M, & Ratner A.J. (2015). Rational Manipulation of mRNA Folding Free Energy Allows Rheostat Control of Pneumolysin Production by Streptococcus pneumoniae. PLoS ONE, 10(3), e0119823.

Publication 2015

LDH cytotoxicity assay

A549 cells Assay Cell Cytotoxicity Epithelial cells Fetal bovine serum Pneumoniae Respiratory Serum Sterile Strains Triton x 100

Corresponding Organization : Columbia University

Other organizations : University of Minho

Top 5 similar protocols

Variable analysis

independent variables

S. pneumoniae strains

dependent variables

LDH cytotoxicity

control variables

A549 respiratory epithelial cells
Minimum essential medium (MEM)
10% fetal bovine serum
Cell monolayers (80–90% confluent in sterile 24-well plates)
Incubation in MEM without serum overnight prior to stimulation

positive control

1% Triton X-100 (100% lysis)

negative control

Vehicle control (0% lysis)

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