Infection of Epithelial Cells With Salmonella enterica - Bacterial Pathogenesis: Methods and Protocols - page 193

10. Phosphate-buffered saline (PBS) without divalent cations: 1.50 g/L dibasic
sodium phosphate (Na 2 HPO 4 ·7H 2 O), 0.2 g/L anhydrous monobasic potassium
phosphate (KH 2 PO 4 ), 8 g/L sodium chloride (NaCl), and 0.2 g/L potassium
chloride (KCl), pH to 7.4.
11. Tissue culture-treated plastic flasks, dishes, and 24-well plates.
12. HeLa cells (human adenocarcinoma cervix epithelial, CCL-2) obtained from the
American Type Culture Collection, Manassas, VA ( see Note 3 ).
13. Hank's-buffered salt solution (HBSS): 0.14 g/L anhydrous calcium chloride
(CaCl 2 ), 0.10 g/L magnesium chloride (MgCl 2 ·H 2 O), 0.10 g/L magnesium
sulfate (MgSO 4 ·7H 2 O), 0.40 g/L potassium chloride (KCl), 0.60 g/L monobasic
potassium phosphate (KH 2 PO 4 ), 0.35 g/L sodium bicarbonate (NaHCO 3 ),
8.0 g/L sodium chloride (NaCl), 48 mg/L anhydrous dibasic sodium phosphate
(Na 2 HPO 4 ), and 1 g/L d-glucose (dextrose).
14. Solubilization buffer: PBS containing 1% Triton X-100 and 0.1% SDS.
15. Sterile glass coverslips, 12-mm diameter (#1).
16. Stock formaldehyde solution (25%) ( see Note 4 ): Dissolve 10 g parafor-
maldehyde in 30 mL water (in capped 50 mL tube). Heat to 75 °C in water bath
with frequent mixing. Add 10 N NaOH to clear (usually 5-20 drops). Add H 2 O
to 40 mL. Store in 1-mL aliquots at -20 °C.
17. Working formaldehyde fixative (2.5%): Thaw aliquot of 25% formaldehyde and
heat to clear. Do not heat formaldehyde above 75 °C. Mix with approximately
6mLofddH 2 O and 1 mL 10× PBS. Adjust pH to 7.2-7.4 with HCl (Use pH
strips, not pH meter). Add ddH 2 Oto10mL.
18. SS-PBS: PBS, 10% (v/v) normal goat serum ( see Note 5 ), and 0.2% (w/v) saponin.
19. Antibodies to lipopolysaccharide (LPS) of S. enterica serovar Typhimurium
(Group B), such as mouse monoclonal antibody, clone 1E6 (BioDesign Interna-
tional, Saco, ME) or rabbit polyclonal antibody, and BBL Salmonella O Group
B antisera (Becton Dickinson, Sparks, MD).
20. Fluorescently conjugated antibodies that have been highly cross-absorbed, such
as Alexa Fluor 488 goat anti-mouse IgG (H + L), Alexa Fluor 568 goat anti-
mouse IgG (H + L) (from Invitrogen™ Corporation), or Cy5 TM -conjugated
Affinipure Goat Anti-Mouse IgG (H + L) (Jackson ImmunoResearch Labora-
tories, Inc., West Grove, PA).
21. Fluorescently conjugated phallotoxins such as Alexa Fluor 488-phalloidin (Invit-
rogen™). Dissolve in methanol (200 U/mL) and store at -20 °C.
22. Mowiol™ mounting media, stock solution: Add 2.4 g of Mowiol™ 4-88 ( see
Note 6 )to6gofglycerol. Stir to mix. Add 6 mL water and leave stirring at
room temperature for several hours. Add 12 mL of 0.2 M Tris-HCl (pH 8.5)
and heat to 50 °C for 10 min with occasional mixing. After Mowiol™ dissolves
(never 100%), clarify by centrifugation at 5000 × g for 15 min. Aliquot into
airtight screw capped 1.5-mL tubes and store at -20 °C. Mowiol™ stock is stable
for a few weeks at room temperature or 4 °C if in an airtight tube. Optional:
Add 1,4,-diazobicyclo-[2.2.2]-octane (DABCO; Sigma-Aldrich) to 2.5% (w/v)
to reduce fading of fluorophores.