polyethersulfone membrane. Store at room temperature. Then
(2) dilute the 50 mM Probenecid stock solution 1:100 in EM
buffer to prepare 500 μM Probenecid solution. Prepare 2.5 μM
Probenecid solution in EM buffer by diluting the 500 μM
solution 1:200 in EM buffer (EM buffer-2.5 μM Probenecid).
And (3) dilute the 1 mM CCF4-AM stock solution 1:6 in
Solution B (provided in Loading kit with CCF4-AM, stored
at room temperature and protected from light) and mix well by
vortexing. And directly add EM buffer-2.5 μM Probenecid to
the previous CC4-AM mix to obtain a substrate loading solution containing 8 μM CCF4-AM. A volume of 25 μL/well is
required.
6. 4% formaldehyde solution (see Subheading 2.16, item 1).
7. DPBS, no calcium, no magnesium, supplemented with 1% FBS
(DPBS-FBS) (see Subheading 2.16, item 5).
2.21 Oxidative Stress
Assays Using Reactive
Oxygen Species (ROS)
Measurement
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): store at À20
C. Prepare
5 mg/mL and 1 mg/mL LPS stock solutions by mixing LPS
solution in endotoxin-free water (provided). Store 20 μL aliquots at À20
C.
4. 100 μg recombinant murine IFNγ (ImmunoTools): Reconstitute the lyophilized IFNγ in 1 mL sterile water to obtain a
100 μg/mL rm. IFNγ stock solution. Store 20 μL aliquots at
À80
C.
5. LPS/ IFNγ solution containing 10 μg/mL LPS and 2 μg/mL
IFNγ: Dilute 1 mg/mL LPS stock solution 1:100 and 100 μg/
mL rm. IFNγ stock solution 1:50 in RPMI-FBS. And secondly
dilute this solution 1:100 in RPMI-FBS to make a working
solution containing 100 ng/mL LPS and 20 ng/mL IFNγ,
respectively.
6. 100 mM NO inducer (L-Arginine) stock solution in deionized
water, 100 μL supplied in ROS-ID NO Detection Kit
(ENZO). Store 10 μL aliquots at À20
C. Avoid repeated
freezing and thawing. Dilute 1:100 in RPMI-FBS to obtain a
1 mM working solution.
7. 2.5 mM CellROX
® Deep Red or Green Oxidative Stress
Reagent in DMSO (Life Technologies). Store at À20
C and
protect from light. For oxidative stress assay, (1) freshly dilute
the 2.5 mM CellROX Reagent 1: 500 in RPMI-FBS to obtain
5 μM CellROX Reagent solution. And (2) prepare 1Â Wash
buffer (provided in the CellROX
® Oxidative Stress kit) by
dilution of 10Â Wash buffer in deionized water.
High-Content Analysis of Host-Pathogen Interactions
661
(2) dilute the 50 mM Probenecid stock solution 1:100 in EM
buffer to prepare 500 μM Probenecid solution. Prepare 2.5 μM
Probenecid solution in EM buffer by diluting the 500 μM
solution 1:200 in EM buffer (EM buffer-2.5 μM Probenecid).
And (3) dilute the 1 mM CCF4-AM stock solution 1:6 in
Solution B (provided in Loading kit with CCF4-AM, stored
at room temperature and protected from light) and mix well by
vortexing. And directly add EM buffer-2.5 μM Probenecid to
the previous CC4-AM mix to obtain a substrate loading solution containing 8 μM CCF4-AM. A volume of 25 μL/well is
required.
6. 4% formaldehyde solution (see Subheading 2.16, item 1).
7. DPBS, no calcium, no magnesium, supplemented with 1% FBS
(DPBS-FBS) (see Subheading 2.16, item 5).
2.21 Oxidative Stress
Assays Using Reactive
Oxygen Species (ROS)
Measurement
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): store at À20
C. Prepare
5 mg/mL and 1 mg/mL LPS stock solutions by mixing LPS
solution in endotoxin-free water (provided). Store 20 μL aliquots at À20
C.
4. 100 μg recombinant murine IFNγ (ImmunoTools): Reconstitute the lyophilized IFNγ in 1 mL sterile water to obtain a
100 μg/mL rm. IFNγ stock solution. Store 20 μL aliquots at
À80
C.
5. LPS/ IFNγ solution containing 10 μg/mL LPS and 2 μg/mL
IFNγ: Dilute 1 mg/mL LPS stock solution 1:100 and 100 μg/
mL rm. IFNγ stock solution 1:50 in RPMI-FBS. And secondly
dilute this solution 1:100 in RPMI-FBS to make a working
solution containing 100 ng/mL LPS and 20 ng/mL IFNγ,
respectively.
6. 100 mM NO inducer (L-Arginine) stock solution in deionized
water, 100 μL supplied in ROS-ID NO Detection Kit
(ENZO). Store 10 μL aliquots at À20
C. Avoid repeated
freezing and thawing. Dilute 1:100 in RPMI-FBS to obtain a
1 mM working solution.
7. 2.5 mM CellROX
® Deep Red or Green Oxidative Stress
Reagent in DMSO (Life Technologies). Store at À20
C and
protect from light. For oxidative stress assay, (1) freshly dilute
the 2.5 mM CellROX Reagent 1: 500 in RPMI-FBS to obtain
5 μM CellROX Reagent solution. And (2) prepare 1Â Wash
buffer (provided in the CellROX
® Oxidative Stress kit) by
dilution of 10Â Wash buffer in deionized water.
High-Content Analysis of Host-Pathogen Interactions
661