4. 1 mg amine-reactive pHrodo™ Red succinimidyl (NHS) Ester
(Life Technologies): resuspend 1 mg of in 1 mL of acetonitrile
solvent to obtain a stock solution of approximately 1.5 mM
pHrodo Red. Make 50 μL aliquots in 1.5-mL microtubes and
dry by use speed vacuum in dark. Store dried aliquots at
À20
C. For phagosomal acidification assay (pHrodo assay),
(1) freshly prepare 50 mL of 100 mM sodium bicarbonate,
pH 8.5. Filter-sterilize through a 0.22 μm cellulose ester membrane. Then (2) resuspend 50 μL of pHrodo Red in 10 μL of
DMSO for a working solution of approximately 7.6 mM
pHrodo Red.
5. CO 2 -independent Medium (Gibco). For phagosomal acidification assay, freshly adjust 10 mL of CO 2 -independent Medium
to a pH range from 4 to 10 (with 100% acetic acid or
5 N NaOH).
6. Mixed and temperature-controlled microtube incubator.
7. 10 mg/mL Hoechst 33342 stock solution (see Subheading
2.16, item 2).
8. DPBS, no calcium, no magnesium, supplemented with 1% FBS
(DPBS-FBS) (see Subheading 2.16, item 5).
2.20 Phagosomal
Vacuole Rupture Assay
1. See items 11–15 under Subheading 2.11.
2. Bafilomycin (SIGMA): store at À20
C. Prepare 0.1 mg/mL
Bafilomycin stock solution (0.16 mM) in DMSO, store 50 μL
aliquots at À20
C for up to 1 year. For phagosomal permeabilization, firstly prepare a 0.16 μM Bafilomycin solution by
diluting the 0.16 mM stock solution 1:1000 in RPMI-FBS.
Secondly, dilute the 0.16 μM Bafilomycin solution 1:8 in
RPMI-FBS to make a 20 nM Bafilomycin working solution
(see Note 13).
3. Heat-killed bacteria (see Subheading 2.18, item 2).
4. 50 mg/mL Amikacin stock solution in PBS (see Subheading
2.12, item 7).
5. 5 mg dried CCF4-AM (Life Technologies): store at À20
C
and protect from light. (1) Prepare 1 mM CCF4-AM stock
solution (named “Solution A” provided in Loading kit with
CCF4-AM) by mixing 5 mg of CCF4-AM in 4.6 mL of DMSO
(provided). Store 50 and 100 μL aliquots at À20
C. (2) Prepare 50 mM Probenecid stock solution in DMSO by suspending 142.68 mg of Probenecid in 10 mL of DMSO. Store
500 μL aliquots at À20
C. For phagosomal vacuole rupture
assay, (1) freshly prepare EM buffer, containing 120 mM
NaCl 2 , 7 mM KCl, 1.8 mM CaCl 2 , 0.8 mM MgCl 2 , 5 mM
glucose and 25 mM Hepes in deionized water. Adjust pH from
5.3 to 7.3 with 5 N NaOH. Filter-sterilize through a 0.22 μm
660
Nathalie Deboosere et al.
(Life Technologies): resuspend 1 mg of in 1 mL of acetonitrile
solvent to obtain a stock solution of approximately 1.5 mM
pHrodo Red. Make 50 μL aliquots in 1.5-mL microtubes and
dry by use speed vacuum in dark. Store dried aliquots at
À20
C. For phagosomal acidification assay (pHrodo assay),
(1) freshly prepare 50 mL of 100 mM sodium bicarbonate,
pH 8.5. Filter-sterilize through a 0.22 μm cellulose ester membrane. Then (2) resuspend 50 μL of pHrodo Red in 10 μL of
DMSO for a working solution of approximately 7.6 mM
pHrodo Red.
5. CO 2 -independent Medium (Gibco). For phagosomal acidification assay, freshly adjust 10 mL of CO 2 -independent Medium
to a pH range from 4 to 10 (with 100% acetic acid or
5 N NaOH).
6. Mixed and temperature-controlled microtube incubator.
7. 10 mg/mL Hoechst 33342 stock solution (see Subheading
2.16, item 2).
8. DPBS, no calcium, no magnesium, supplemented with 1% FBS
(DPBS-FBS) (see Subheading 2.16, item 5).
2.20 Phagosomal
Vacuole Rupture Assay
1. See items 11–15 under Subheading 2.11.
2. Bafilomycin (SIGMA): store at À20
C. Prepare 0.1 mg/mL
Bafilomycin stock solution (0.16 mM) in DMSO, store 50 μL
aliquots at À20
C for up to 1 year. For phagosomal permeabilization, firstly prepare a 0.16 μM Bafilomycin solution by
diluting the 0.16 mM stock solution 1:1000 in RPMI-FBS.
Secondly, dilute the 0.16 μM Bafilomycin solution 1:8 in
RPMI-FBS to make a 20 nM Bafilomycin working solution
(see Note 13).
3. Heat-killed bacteria (see Subheading 2.18, item 2).
4. 50 mg/mL Amikacin stock solution in PBS (see Subheading
2.12, item 7).
5. 5 mg dried CCF4-AM (Life Technologies): store at À20
C
and protect from light. (1) Prepare 1 mM CCF4-AM stock
solution (named “Solution A” provided in Loading kit with
CCF4-AM) by mixing 5 mg of CCF4-AM in 4.6 mL of DMSO
(provided). Store 50 and 100 μL aliquots at À20
C. (2) Prepare 50 mM Probenecid stock solution in DMSO by suspending 142.68 mg of Probenecid in 10 mL of DMSO. Store
500 μL aliquots at À20
C. For phagosomal vacuole rupture
assay, (1) freshly prepare EM buffer, containing 120 mM
NaCl 2 , 7 mM KCl, 1.8 mM CaCl 2 , 0.8 mM MgCl 2 , 5 mM
glucose and 25 mM Hepes in deionized water. Adjust pH from
5.3 to 7.3 with 5 N NaOH. Filter-sterilize through a 0.22 μm
660
Nathalie Deboosere et al.