5. 1 mg/mL DAPI stock solution (see Subheading 2.16, item 3).
6. DPBS, no calcium, no magnesium, supplemented with 1% FBS
(DPBS-FBS) (see Subheading 2.16, item 5).
2.24 Lipid Body
Accumulation Assay
1. See items 11–15 under Subheading 2.11.
2. 50 mg/mL Amikacin stock solution in PBS (see Subheading
2.12, item 7).
3. LPS-EB Ultrapure (InvivoGen) (see Subheading 2.21, item 3).
4. 100 μg recombinant murine IFNγ (ImmunoTools) (see Subheading 2.21, item 4).
5. LPS/IFNγ solution containing 100 ng/mL LPS and 20 ng/
mL IFNγ (see Subheading 2.21, item 5).
6. Oleic acid. Prepare 6 mM Oleic acid stock solution. For lipid
body accumulation assays in bone marrow dendritic cells
(BMDC), prepare a 60 μM Oleic acid solution by diluting the
stock solution 1:1000 in RPMI-FBS.
7. 4% formaldehyde solution (see Subheading 2.16, item 1).
8. 10 mg/mL Hoechst 33342 stock solution (see Subheading
2.16, item 2).
9. HCS LipidTOX™ Far-Red or Green Neutral Lipid Stain solution (Invitrogen) (corresponding to 1000Â solution in
DMSO). Store at À20
C and protect from light. For lipid
body accumulation assay, freshly dilute the 1000Â LipidTOX
neutral lipid stain 1:2000 in PBS to make a 0.5Â solution (see
Note 14).
2.25 Image
Acquisition
and Analysis
1. Cell nuclei and cytoplasm dyes: SYTO 60 Red Fluorescent
Nucleic Acid Stain, Hoechst, DAPI, CellMask Blue Stain,
CellMask Deep Red Plasma Membrane stain (see Subheadings
2.12, 2.15, 2.16, and 2.17).
2. Organelle labeling: Alexa Fluor 660 Phalloidin Stain, LysoTracker Green Dye, LysoTracker Red Dye, CCF4-AM, LipidTox Green Neutral Lipid Stain, LipidTox Far-Red Neutral
Lipid Stain, (see Subheadings 2.17, 2.18, 2.20, and 2.24).
3. Molecules or pH change detection: CellROX
® Green Oxidative Stress Reagent, ROS-ID
® Red NO Detection Reagent,
FluoZin™-3 AM cell permeant, Amine-reactive pHrodo™
Red NHS Ester (see Subheadings 2.19, 2.21–2.23).
4. Automated laser-scanning confocal microscope: Opera (Perkin
Elmer) or similar instrument adapted for 384-well plates,
equipped with 405 nm, 488 nm, 561 nm, and 640 nm excitation lasers as well as 20Â and 60Â objectives.
5. Image analysis software (see Note 15).
High-Content Analysis of Host-Pathogen Interactions
663
6. DPBS, no calcium, no magnesium, supplemented with 1% FBS
(DPBS-FBS) (see Subheading 2.16, item 5).
2.24 Lipid Body
Accumulation Assay
1. See items 11–15 under Subheading 2.11.
2. 50 mg/mL Amikacin stock solution in PBS (see Subheading
2.12, item 7).
3. LPS-EB Ultrapure (InvivoGen) (see Subheading 2.21, item 3).
4. 100 μg recombinant murine IFNγ (ImmunoTools) (see Subheading 2.21, item 4).
5. LPS/IFNγ solution containing 100 ng/mL LPS and 20 ng/
mL IFNγ (see Subheading 2.21, item 5).
6. Oleic acid. Prepare 6 mM Oleic acid stock solution. For lipid
body accumulation assays in bone marrow dendritic cells
(BMDC), prepare a 60 μM Oleic acid solution by diluting the
stock solution 1:1000 in RPMI-FBS.
7. 4% formaldehyde solution (see Subheading 2.16, item 1).
8. 10 mg/mL Hoechst 33342 stock solution (see Subheading
2.16, item 2).
9. HCS LipidTOX™ Far-Red or Green Neutral Lipid Stain solution (Invitrogen) (corresponding to 1000Â solution in
DMSO). Store at À20
C and protect from light. For lipid
body accumulation assay, freshly dilute the 1000Â LipidTOX
neutral lipid stain 1:2000 in PBS to make a 0.5Â solution (see
Note 14).
2.25 Image
Acquisition
and Analysis
1. Cell nuclei and cytoplasm dyes: SYTO 60 Red Fluorescent
Nucleic Acid Stain, Hoechst, DAPI, CellMask Blue Stain,
CellMask Deep Red Plasma Membrane stain (see Subheadings
2.12, 2.15, 2.16, and 2.17).
2. Organelle labeling: Alexa Fluor 660 Phalloidin Stain, LysoTracker Green Dye, LysoTracker Red Dye, CCF4-AM, LipidTox Green Neutral Lipid Stain, LipidTox Far-Red Neutral
Lipid Stain, (see Subheadings 2.17, 2.18, 2.20, and 2.24).
3. Molecules or pH change detection: CellROX
® Green Oxidative Stress Reagent, ROS-ID
® Red NO Detection Reagent,
FluoZin™-3 AM cell permeant, Amine-reactive pHrodo™
Red NHS Ester (see Subheadings 2.19, 2.21–2.23).
4. Automated laser-scanning confocal microscope: Opera (Perkin
Elmer) or similar instrument adapted for 384-well plates,
equipped with 405 nm, 488 nm, 561 nm, and 640 nm excitation lasers as well as 20Â and 60Â objectives.
5. Image analysis software (see Note 15).
High-Content Analysis of Host-Pathogen Interactions
663