Table 1.
Screening of Multi-TLR Signaling in PAMP-Treated or Bacteria-Infected IMMs
| Step | Parameter | Value | Description |
|---|---|---|---|
| Cell culture and ligand treatment (1–5) | |||
| 1 | Plate IMMs | 200 μL | 0.5 × 105 cells/mL; 96-well plates |
| 2 | Incubate cells | 24 h | 37°C, 5% CO2, 95% humidity |
| 3 | Aspirate and incubate with fresh medium | 2 h | cDMEM at 37°C |
| 4 | Treat cells with PAMPs | 200 μL, 0–24 h | Aspirate medium from appropriate wells and add fresh medium containing PAMPs at desired concentration. Incubate at 37°C, 5% CO2, 95% humidity for the desired length of time |
| 5 | Aspirate and wash (2×) with PBS | 100 μL | Prewarmed PBS at 37°C without Ca2+/Mg2+ (100 μL/well, 2×) |
| Fixation and staining (6–14) | |||
| 6 | Fixation | 100 μL, 20 min | Aspirate and add 4% PFA in PBS without Ca2+/Mg2+, 37°C |
| 7 | Blocking and permeabilization | 100 μL | Aspirate and add 5% (w/v) BSA in 0.1% (v/v) Tween 20 in 1× PBS. At room temperature for at least 1 h or overnight at 4°C |
| 8 | Primary antibody staining | 100 μL | Use appropriate dilutions of primary antibodies prepared in blocking solution described above. Incubate on rocker overnight at 4°C |
| 9 | Wash and aspirate (3–5×) | 100 μL | Wash with blocking solution for at least 5 min per wash on rocker at room temperature. Aspirate at end of each wash cycle |
| 10 | Secondary antibody staining | 100 μL | At room temperature for at least 1 h on a rocker. The plate needs to be protected from light from this point onward |
| 11 | Wash and aspirate (3–5×) | 100 μL | Repeat as described in step 9 |
| 12 | Nuclear staining | 100 μL, 20 min | Add four drops of Hoechst 33342 in 20 mL of blocking solution and use this solution for nuclear staining. Incubate on rocker for ∼20 min |
| 13 | Wash and aspirate (1–2×) | 100 μL | Repeat as described in step 11. At the end of the wash cycle, add 200 μL of blocking solution per well |
| 14 | Seal plates | Use clear film to seal the plates | |
| Image acquisition and data analysis (15–17) | |||
| 15 | Acquire images | 20×, 0.45 NA objective | Autofocus was applied using the DAPI channel (Ch1). Up to three channels—green, red, and far red—are imaged in sequence. Imaging was performed using a Cell Insight NXT |
| 16 | Assay readout | The general intensity measurement bioapplication was used to count Ch2 (green stained) objects located within circle (nucleus) and ring (cytoplasm) masks defined by Ch1 objects (Hoechst stained) | |
| 17 | Analysis | Ratios of nuclear (circle) to cytoplasmic (ring) Ch2 intensities are calculated for all objects defined by Ch1. The basal mean +2SD values are calculated for each treatment. Mean intensities are then calculated for all cells above basal mean +2SD. These mean values are then normalized to maximum values in each treatment | |
Step Notes
1. These cells are good for setting up ligand treatment for imaging up to 3 days after which point they become overconfluent.
7. Blocking solution is made in bulk. It is filter sterilized and stored at 4°C. Filtration reduces particles that can cause nonspecific background staining and the sterility helps keep the plates good for imaging for over 4 months when stored in the dark at 4°C.
10–14. Plates should be protected from light.
16. General intensity measurement bioapplication from Thermo Scientific™ HCS Studio™ Cell Analysis Software.
BSA, bovine serum albumin; HCS, high-content screening; IMM, immortalized murine macrophage; PAMP, pathogen-associated molecular pattern; PBS, phosphate-buffered saline; PFA, paraformaldehyde.