2.2 Cell Monolayer
Resistance
1. Electrode and transepithelial/transendothelial electrical resistance (TEER) measurement device (see Note 2).
2. Complete growth medium.
3. Sterile PBS.
4. 10% trypan blue solution: 100 μL trypan blue, 900 μL water.
2.3 Infection
1. EMJH base: 2.3 g of Difco Leptospira Medium Base EMJH
(Becton Dickinson) in 890 mL of water + 0.01% glycerol,
pH 7.4.
2. EMJH supplement: 1% bovine serum albumin (BSA), 0.005%
Thiamine hydrochloride, 0.01% CaCl 2 , 0.01% MgCl 2 , 0.004%
ZnSO 4 , 0.0002% vitamin B12, 0.05% FeSO 4 , 1.25% Tween
80, 0.0003% of MgSO 4 in 100 mL of water, pH 7.4.
3. Enriched EMJH supplement: 1% of lactalbumin hydrolysate,
0.04% of sodium pyruvate, 1KU superoxide dismutase, and
10 mL of heat-inactivated rabbit serum in 100 mL of EMJH
supplement.
4. Leptospira EMJH Medium: 890 mL of EMJH base + 110 mL
of Enriched EMJH supplement.
5. Sterile PBS.
6. Leptospira sp. cultures in mid-log growth.
7. Petroff-Hausser counting chamber.
8. Trypsin/EDTA solution.
9. Complete growth medium.
10. EMJH/DMEM medium: 1:2 v/v ratio of DMEM and Leptospira EMJH media.
3 Methods
Follow strict aseptic technique to perform every procedure.
Eukaryotic cells and Leptospira strains must be handled in a Class
II biosafety cabinet to prevent foreign contamination and cross
contamination. All surfaces should be cleaned with 70% ethanol
before and after use. Class II biosafety cabinet should be treated
with UV light for 20 min before and after use.
3.1 Preparation of
Leptospira EMJH
Medium [15, 16]
1. Prepare stock solutions that can be kept at À20
C for up to
1 month: 10% glycerol, 10KU superoxide dismutase (SOD),
0.4% ZnSO 4 , 1% CaCl 2 , 1% MgCl 2 , 0.5% thiamine hydrochloride, 0.02% vitamin B12, and 0.3% MgSO 4 .
2. Prepare fresh stock solutions: 0.5% FeSO 4 and 10% Tween 80.
3. To prepare 100 mL of EMJH supplement, dissolve 10 g of
bovine serum albumin (BSA) into 50 mL of distilled water, and
stir slowly overnight to avoid foam. Once the BSA is fully
Cell Monolayer Translocation Assay
163
Resistance
1. Electrode and transepithelial/transendothelial electrical resistance (TEER) measurement device (see Note 2).
2. Complete growth medium.
3. Sterile PBS.
4. 10% trypan blue solution: 100 μL trypan blue, 900 μL water.
2.3 Infection
1. EMJH base: 2.3 g of Difco Leptospira Medium Base EMJH
(Becton Dickinson) in 890 mL of water + 0.01% glycerol,
pH 7.4.
2. EMJH supplement: 1% bovine serum albumin (BSA), 0.005%
Thiamine hydrochloride, 0.01% CaCl 2 , 0.01% MgCl 2 , 0.004%
ZnSO 4 , 0.0002% vitamin B12, 0.05% FeSO 4 , 1.25% Tween
80, 0.0003% of MgSO 4 in 100 mL of water, pH 7.4.
3. Enriched EMJH supplement: 1% of lactalbumin hydrolysate,
0.04% of sodium pyruvate, 1KU superoxide dismutase, and
10 mL of heat-inactivated rabbit serum in 100 mL of EMJH
supplement.
4. Leptospira EMJH Medium: 890 mL of EMJH base + 110 mL
of Enriched EMJH supplement.
5. Sterile PBS.
6. Leptospira sp. cultures in mid-log growth.
7. Petroff-Hausser counting chamber.
8. Trypsin/EDTA solution.
9. Complete growth medium.
10. EMJH/DMEM medium: 1:2 v/v ratio of DMEM and Leptospira EMJH media.
3 Methods
Follow strict aseptic technique to perform every procedure.
Eukaryotic cells and Leptospira strains must be handled in a Class
II biosafety cabinet to prevent foreign contamination and cross
contamination. All surfaces should be cleaned with 70% ethanol
before and after use. Class II biosafety cabinet should be treated
with UV light for 20 min before and after use.
3.1 Preparation of
Leptospira EMJH
Medium [15, 16]
1. Prepare stock solutions that can be kept at À20
C for up to
1 month: 10% glycerol, 10KU superoxide dismutase (SOD),
0.4% ZnSO 4 , 1% CaCl 2 , 1% MgCl 2 , 0.5% thiamine hydrochloride, 0.02% vitamin B12, and 0.3% MgSO 4 .
2. Prepare fresh stock solutions: 0.5% FeSO 4 and 10% Tween 80.
3. To prepare 100 mL of EMJH supplement, dissolve 10 g of
bovine serum albumin (BSA) into 50 mL of distilled water, and
stir slowly overnight to avoid foam. Once the BSA is fully
Cell Monolayer Translocation Assay
163