8. 4% formaldehyde solution (see Subheading 2.16, item 1).
9. 10 mg/mL Hoechst 33342 stock solution (see Subheading
2.16, item 2).
10. DPBS, no calcium, no magnesium, supplemented with 1% FBS
(DPBS-FBS) (see Subheading 2.16, item 5).
2.22 Oxidative Stress
Assays Using
Intracellular Nitric
Oxide
(NO) Detection Assay
1. ROS-ID
® NO Detection Reagent (Red) in Dimethylformamide (DMF) (ENZO). Store 6 μL aliquots at À20
C and
protect from light. Avoid repeated freezing and thawing. For
oxidative stress assay, freshly dilute the probe 1:400 by adding
2.5 μL of the reagent per 1 mL of pre-warmed (37
C) RPMIFBS.
2. 10 mg/mL Hoechst 33342 stock solution (see Subheading
2.16, item 2).
3. Lyophilized NO Scavenger (c-PTIO), 400 nmoles supplied in
ROS-ID NO Detection Kit (ENZO). Reconstitute in 100 μL
of anhydrous 100% Dimethylformamide (DMF) to make a
4 mM stock concentration. Store 10 μL aliquots at À20
C.
For oxidative stress assay, dilute the 4 mM stock solution from
1:50 up to 1:200 in RPMI-FBS to obtain the recommended
final concentration from 20 up to 80 μM.
4. 5 mM NO Inducer (L-Arginine): dilute the 5 mM stock solution 1:5 in RPMI-FBS to obtain final concentration of 1 mM.
5. Complete RPMI medium without red phenol (RPMI-FBS
without red phenol): add 50 mL of FBS to a 500 mL bottle
of RPMI 1640 medium supplemented with 2 mM glutamine.
Store at 4
C for up to 1 month.
2.23 Zinc
Poisoning Assay
1. See items 11–15 under Subheading 2.11.
2. 4% formaldehyde solution (see Subheading 2.16, item 1).
3. FluoZin™-3, AM, cell permeant (Invitrogen). (1) Reconstitute
100 μg in high-quality anhydrous DMSO to obtain concentrations of 2 mM. Store 10 μL aliquots at À20
C and protect
from light. Avoid repeated freezing and thawing. For zinc
poisoning assay, freshly dilute the probe 1:1000 in DPBS to
obtain 2 μM FluoZin-3.
4. Indicator salts, 500 μg provided in FluoZin™-3, AM, cell
permeant (Invitrogen). Prepare stock solutions of watersoluble indicator salts by diluting in distilled water to obtain
1–10 mM indicator solutions. Store 50 μL aliquots at À20
C
and protect from light. For zinc poisoning assay, freshly dilute
the salt 1:100 in pre-warmed (37
C) RPMI-FBS to obtain a
final intracellular indicator concentration of 10–100 μM.
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Nathalie Deboosere et al.
9. 10 mg/mL Hoechst 33342 stock solution (see Subheading
2.16, item 2).
10. DPBS, no calcium, no magnesium, supplemented with 1% FBS
(DPBS-FBS) (see Subheading 2.16, item 5).
2.22 Oxidative Stress
Assays Using
Intracellular Nitric
Oxide
(NO) Detection Assay
1. ROS-ID
® NO Detection Reagent (Red) in Dimethylformamide (DMF) (ENZO). Store 6 μL aliquots at À20
C and
protect from light. Avoid repeated freezing and thawing. For
oxidative stress assay, freshly dilute the probe 1:400 by adding
2.5 μL of the reagent per 1 mL of pre-warmed (37
C) RPMIFBS.
2. 10 mg/mL Hoechst 33342 stock solution (see Subheading
2.16, item 2).
3. Lyophilized NO Scavenger (c-PTIO), 400 nmoles supplied in
ROS-ID NO Detection Kit (ENZO). Reconstitute in 100 μL
of anhydrous 100% Dimethylformamide (DMF) to make a
4 mM stock concentration. Store 10 μL aliquots at À20
C.
For oxidative stress assay, dilute the 4 mM stock solution from
1:50 up to 1:200 in RPMI-FBS to obtain the recommended
final concentration from 20 up to 80 μM.
4. 5 mM NO Inducer (L-Arginine): dilute the 5 mM stock solution 1:5 in RPMI-FBS to obtain final concentration of 1 mM.
5. Complete RPMI medium without red phenol (RPMI-FBS
without red phenol): add 50 mL of FBS to a 500 mL bottle
of RPMI 1640 medium supplemented with 2 mM glutamine.
Store at 4
C for up to 1 month.
2.23 Zinc
Poisoning Assay
1. See items 11–15 under Subheading 2.11.
2. 4% formaldehyde solution (see Subheading 2.16, item 1).
3. FluoZin™-3, AM, cell permeant (Invitrogen). (1) Reconstitute
100 μg in high-quality anhydrous DMSO to obtain concentrations of 2 mM. Store 10 μL aliquots at À20
C and protect
from light. Avoid repeated freezing and thawing. For zinc
poisoning assay, freshly dilute the probe 1:1000 in DPBS to
obtain 2 μM FluoZin-3.
4. Indicator salts, 500 μg provided in FluoZin™-3, AM, cell
permeant (Invitrogen). Prepare stock solutions of watersoluble indicator salts by diluting in distilled water to obtain
1–10 mM indicator solutions. Store 50 μL aliquots at À20
C
and protect from light. For zinc poisoning assay, freshly dilute
the salt 1:100 in pre-warmed (37
C) RPMI-FBS to obtain a
final intracellular indicator concentration of 10–100 μM.
662
Nathalie Deboosere et al.