Murine bone marrow-derived macrophages were stimulated in vitro with the three TLR agonists LPS, Pam3CSK4 and poly I:C were stained with phospho-specific antibodies for a panel of signaling molecules and subjected to multi-parameter cytofluorometric analysis.
This data set is provided courtesy of the Cell Signaling Core at Stanford (Drew Hotson, Fred Elfman, and the laboratory of Garry Nolan) and the Genomics Core at ISB (Kathleen Kennedy, Elizabeth Gold, and the laboratory of Alan Aderem). Murine bone marrow-derived macrophages stimulated in vitro with the three TLR agonists LPS, Pam3CSK4 and poly I:C have been stained with phospho-specific antibodies for a panel of signaling molecules (using the Alexa fluor 647, or Ax647) and subjected to multi-parameter cytofluorometric analysis using the temporal barcoding technique described in Krutzik PO et al., Nat Methods 3(5), 361-8 (2006). The temporal barcoding was accomplished by staining cells in unique combinations of concentrations of PacBlu and PacOrange succimidyl esters (in PBS to MeOH ratio of 2:1) at various time points post-stimulation, as shown:
| Time Point | PacBlu | PacOrange |
| 0 min | 0.33 μg/ml | none |
| 5 min | 2 μg/ml | none |
| 10 min | none | 0.33 μg/ml |
| 20 min | 2 μg/ml | 0.33 μg/ml |
| 40 min | none | 2 μg/ml |
| 60 min | 0.33 μg/ml | 2 μg/ml |
A time point of 0 indicates no stimulation. Please note that the barcoding intensities must be compensated for spectral cross-talk between PacBlu and PacOrange (The corrected PacOrange intensity value should be computed as the measured PacOrange intensity value minus 0.29 times the measured PacBlue intensity value. The bleed-through of PacOrange into the PacBlue intensity measurement is less than 5%. Neither of the barcoding stains affects the Ax647 intensity measurement). Cells were recombined and stained with phospho-specific antibodies (for signaling molecules as indicated below) labeled with Ax647, with μl / 100 μl stain volume.
Download the phospho-FACS data file archive: