Chemi touch imaging system

Lamina propria and mesenteric lymph node cells were collected from naïve and infected MyD88^+/+ and MyD88^-/-IL12-p40 eYFP mice. The cells were cultured ex vivo in cDMEM and supernatants were collected after 72 hours. Cytokines and chemokines were quantified simultaneously in samples using the Proteome Profiler Mouse XL Cytokine Array kit following the manufacturer’s protocol (R&D Systems, Minneapolis, MN). Briefly, the proteome array blots were hydrated and blocked, incubated with the supernatant samples overnight, and probed with biotinylated detection antibodies and streptavidin-horseradish peroxidase. After application of the substrate (3,3’,5,5’-Tetramethylbenzidine), blots were visualized on a Chemi Touch Imaging System (Bio-Rad), and Image J software was used to quantitate the mean pixel intensities of each cytokine and chemokine using background subtraction of control spots containing no cytokine/chemokine antibody.

Cytokine and chemokine expression was measured using the Murine Proteome Profiler Mouse XL Cytokine Array (R&D Systems, Minneapolis, MN). To obtain samples, C57BL/6 mice were i. p. inoculated with either cps1-1 or cps1-1:Δgra24 (10⁶ tachyzoites per mouse). Four days post infection the peritoneal exudate cells were collected by filling the peritoneal cavity with sterile PBS then using a 21 Ga needle to collect the cell suspension. The PECs were plated in cDMEM at 2 x 10⁶ cells per well and were incubated for 72 hr. Following incubation, the supernatants were collected and the Murine Proteome Profiler Mouse XL Cytokine Array was used according to the manufacturer’s directions. In brief, nitrocellulose membranes were blocked for 1 hr then sample supernatants were added to the membranes. After overnight incubation, membranes were washed and a detection antibody cocktail (R&D Systems) was added. The membranes were incubated with the detection antibody for 1 hr, washed, and streptavidin-horseradish peroxidase was added. After 30 min incubation, membranes were washed three times (10 min per wash) and spots visualized by addition of enhanced chemiluminescence reagent. The membranes were imaged on a Chemi Touch Imaging System (Bio-Rad), and semi-quantitative analysis was accomplished using ImageJ software.

30 μg of total protein for input or 45 μl of pulldown supernatant was loaded into 4–20% Mini-PROTEAN® TGX Stain-Free™ Protein Gels. Total protein was assessed through stain-free imaging on a Biorad ChemiTouch Imaging System, which allows total protein loaded into the well to be used as the loading control. Protein was transferred onto the PVDF membrane (Biorad, 162–0255) using the Trans-Blot Turbo Transfer System (Biorad 1704150). Membranes were blocked with 5% milk in 0.1% TBST for 1 hour and probed with one of the following antibodies at the designated dilution overnight at 4°C. 1:3000 Monoclonal ANTI-FLAG® M2 antibody (Sigma, F1804, Lot. SLBK1346V), 1:1000 anti-His HRP antibody (GenScript, A00612, Lot. 19K001984), 1:1000 anti-HA-Tag (C29F4) Rabbit mAb (Cell Signaling, 37245, Lot. 8), or 1:1000 anti-Human/Mouse/Rat PRL-3 Antibody (R&D Systems, MAB3219, Lot. WXH0419091). Following three washes with 0.1% TBST, secondary HRP-conjugated 1:2500 anti-mouse IgG antibody (Cell Signaling, 7076S, Lot. 33) or 1:5000 anti-Rabbit IgG HRP Linked F(ab′)2 (Sigma, NA9340V, Lot. 17065618) was added for 1 hour and membranes were imaged using Clarity Western ECL Substrate (Biorad, 1705061).