2.17 Staining of Cell
Nucleus, Cytoplasm,
and/or F-Actin
on Fixed and
Permeabilized Cells
1. 4% formaldehyde solution (see Subheading 2.16, item 1).
2. 0.1% w/v Triton X-100 solution: add 0.1 g Triton X-100 to
100 mL of PBS. Solubilize by agitation at 37
C and filtersterilize through a 0.22 μm polyethersulfone membrane. Store
at 4
C for several months.
3. 10 mg/mL Hoechst 33342 stock solution (see Subheading
2.16, item 2).
4. HCS CellMask™ Blue Stain (Invitrogen). Reconstitute HCS
CellMask™ Stain in 25 μL DMSO (supplied) to make a
10 mg/mL stock solution. Store at À20
C and protect from
light. For appropriate assay, freshly dilute the stock solution
1:5000 in DPBS to obtain 2 μg/mL CellMask Blue stain
solution.
5. 100 nmoles Alexa Fluor 660 Phalloidin (Life Technologies,
300 Units). Reconstitute Alexa Fluor 660 Phalloidin in
1.5 mL of methanol to obtain an equivalent to approximately
66 μM Alexa Fluor 660 Phalloidin stock solution. Store at
À20
C and protect from light. For appropriate assay, freshly
dilute Alexa Fluor 660 Phalloidin stock solution 1:40 in DPBS
to obtain 1.65 μM working solution.
6. DPBS, no calcium, no magnesium, supplemented with 1% FBS
(DPBS-FBS) (see Subheading 2.16, item 5).
2.18 Phagosomal
Acidification Assay
Using LysoTracker
Reagent
1. See items 11–15 under Subheading 2.11.
2. Heat-killed bacteria. Heat 1 mL of the bacterial suspension
corresponding to the tested MOI, used for infection, at
95
C for 30 min.
3. 50 mg/mL Amikacin stock solution in PBS (see Subheading
2.12, item 7).
4. 1 mM LysoTracker™ Green DND-26 and Red DND-99 dyes
(Life Technologies) in DMSO: store at À20
C and protect
from light. For phagosomal acidification assay, freshly dilute
the 1 mM LysoTracker dye 1:1000 in appropriate complete
medium to obtain 1 μM LysoTracker solution (see Note 12).
5. 4% formaldehyde solution (see Subheading 2.16, item 1).
6. 10 mg/mL Hoechst 33342 stock solution (see Subheading
2.16, item 2).
7. DPBS, no calcium, no magnesium, supplemented with 1% FBS
(DPBS-FBS) (see Subheading 2.16, item 5).
2.19 Phagosomal
Acidification Assay
Using pHrodo Reagent
1. See items 11–15 under Subheading 2.11.
2. Laboratory centrifuge to achieve 3750 Â g.
3. 100 mM sodium bicarbonate buffer, pH 8.3.
High-Content Analysis of Host-Pathogen Interactions
659
Nucleus, Cytoplasm,
and/or F-Actin
on Fixed and
Permeabilized Cells
1. 4% formaldehyde solution (see Subheading 2.16, item 1).
2. 0.1% w/v Triton X-100 solution: add 0.1 g Triton X-100 to
100 mL of PBS. Solubilize by agitation at 37
C and filtersterilize through a 0.22 μm polyethersulfone membrane. Store
at 4
C for several months.
3. 10 mg/mL Hoechst 33342 stock solution (see Subheading
2.16, item 2).
4. HCS CellMask™ Blue Stain (Invitrogen). Reconstitute HCS
CellMask™ Stain in 25 μL DMSO (supplied) to make a
10 mg/mL stock solution. Store at À20
C and protect from
light. For appropriate assay, freshly dilute the stock solution
1:5000 in DPBS to obtain 2 μg/mL CellMask Blue stain
solution.
5. 100 nmoles Alexa Fluor 660 Phalloidin (Life Technologies,
300 Units). Reconstitute Alexa Fluor 660 Phalloidin in
1.5 mL of methanol to obtain an equivalent to approximately
66 μM Alexa Fluor 660 Phalloidin stock solution. Store at
À20
C and protect from light. For appropriate assay, freshly
dilute Alexa Fluor 660 Phalloidin stock solution 1:40 in DPBS
to obtain 1.65 μM working solution.
6. DPBS, no calcium, no magnesium, supplemented with 1% FBS
(DPBS-FBS) (see Subheading 2.16, item 5).
2.18 Phagosomal
Acidification Assay
Using LysoTracker
Reagent
1. See items 11–15 under Subheading 2.11.
2. Heat-killed bacteria. Heat 1 mL of the bacterial suspension
corresponding to the tested MOI, used for infection, at
95
C for 30 min.
3. 50 mg/mL Amikacin stock solution in PBS (see Subheading
2.12, item 7).
4. 1 mM LysoTracker™ Green DND-26 and Red DND-99 dyes
(Life Technologies) in DMSO: store at À20
C and protect
from light. For phagosomal acidification assay, freshly dilute
the 1 mM LysoTracker dye 1:1000 in appropriate complete
medium to obtain 1 μM LysoTracker solution (see Note 12).
5. 4% formaldehyde solution (see Subheading 2.16, item 1).
6. 10 mg/mL Hoechst 33342 stock solution (see Subheading
2.16, item 2).
7. DPBS, no calcium, no magnesium, supplemented with 1% FBS
(DPBS-FBS) (see Subheading 2.16, item 5).
2.19 Phagosomal
Acidification Assay
Using pHrodo Reagent
1. See items 11–15 under Subheading 2.11.
2. Laboratory centrifuge to achieve 3750 Â g.
3. 100 mM sodium bicarbonate buffer, pH 8.3.
High-Content Analysis of Host-Pathogen Interactions
659