Details of TLR Signaling Example
In Vitro Stimulation of Bone Marrow-Derived Macrophages by TLR Agonists over a Time Course as Measured with Multiple Panels of Phospho-protein Stains
In order to study the role of the protein MyD88, wild-type (wt) or MyD88 KO-derived bone marrow derived macrophages (BMDM) were stimulated in vitro by TLR agonists: PAM 3, Poly IC, or LPS, exposed to cells for: 0, 5, 10, 20, 40 or 60 minutes, then stained with a panel of antibodies (Abs.) targeting the phosphorylated variants of signaling intermediates postulated to be downstream of MyD88. Prior to barcoding, cells were divided by mouse genotype/phenotype (wt or MyD88 KO) and in vitro stimuli ( PAM 3 / Poly IC / LPS) in parameter groups I and II (below), generating a total of 6 primary groupings:wt/PAM 3; wt/Poly IC; wt/LPS; KO/PAM 3; KO/Poly IC; KO/LPS. The bone marrow-derived macrophages (BMDM), stimulated in vitro, were fixed (1.6% PFA), and permeabilized (cold MeOH), following in vitro stimulations.
A labeling matrix containing Pac Orange succinimidyl ester (PO) by Pac Blue succinimidyl ester (PB), each: 0, 0.333 µg, or 2 µg/ total staining volume, allows for the “pre-labeling” of as many as 8 data points prior to staining for phospho-flow markers (for example: in vitro stimuli- time course data points: 0, 5,10, 20, 40, or 60 minutes). Other parameters that are well suited for secondary grouping: Dose response data points for a single stimulus or combinations of a stimulus with a dose range of inhibitors or cocktails. Simultaneous staining of pooled, coded samples saves reagents while improving the precision of the measurement of subtle changes.
Time course data points were organized by secondary groupings that are optimally suited for barcoding with the FCB method such as in a PB by PO matrix (parameter group IV, below). Preparation of bar-coding stocks (and phospho-flow antibody staining stocks) in batch maximizes the consistency of their labeling. Note: PBS without albumin or serum is used and labeling is done using 106 BMDM immediately post-permeabilization in 100ul MeOH. To this volume, PB and/or PO dyes are added in 200ul of PBS for a final volume of 300ul, to obtain the final concentration indicated. Next, individually labeled time course data points are pooled, resuspended to a total of 1100 ul (100ul/each stain), and distributed to 11 tubes (or wells) for phospho-flow staining. Thus, different time course data points within each of the primary groups (wt/LPS, for example) can be “separated” during post-flow cytometer data analysis (FlowJo or Stanford CytoBank) using the forward deconvolution method developed in the Nolan lab.
Parameter Data Group III, the staining of phospho-flow proteins, generates a final level of primary grouping. Unconjugated rabbit or mouse Abs (also directly conjugated Ax647-A primary Abs.) are added separately to each staining aliquot (100ul ~ 900K cells), stained, washed, and incubated with Goat anti rabbit (or mouse) secondary Abs. (1:5K), washed and resuspended in 50ul Staining Medium (0.5% BSA or 1% FBS w/ .02% NaN3) for FACS analysis. The feasibility of adding additional Abs (mouse, if rabbit has been used, or visa versa) directly conjugated to Ax488-A, or secondary Abs. conjugated to Ax488-A (and perhaps additional conjugates, if possible) for multiple stainings at this step is under investigation. The selection of Abs. for staining in Group III is dictated by the relevant experimental issues for a given study. For this study, three panels of phospho-flow markers with relevance to TLR signaling were selected. NOTE: pMAPKAPK2 provides a consistent positive control in these systems and is therefore included in each panel. Such a positive control is suggested when assembling staining panels.
REFERENCES
1. Krutzik P.O., Crane J.M., Clutter M.R., Nolan G.P. (2008). High-content single-cell drug screening with phosphospecific flow cytometry. Nat Chem Biol. Feb;4(2):132-42. Epub 2007 Dec 23.
