dissolved, add 1 mL of the following stock solutions: thiamine,
CaCl 2 , MgCl 2 , ZnSO 4 , and vitamin B12. Also add 10 mL of
FeSO 4 , 12.5 mL of Tween 80, and 0.1 mL of MgSO 4 stock
solutions. Adjust pH to 7.4 with NaOH and adjust volume to
100 mL with water. The EMJH supplement can be stored at
À20
C indefinitely until use.
4. For 1 L of EMJH medium, dissolve 2.3 g of Difco Leptospira
Medium Base EMJH in 890 mL of water. Add 0.9 mL of 10%
glycerol stock solution and adjust pH to 7.4 with HCl.
5. Thaw 100 mL of EMJH supplement and add 1 g of lactalbumin hydrolysate, 0.04 g of sodium pyruvate, 100 μL of SOD
stock solution, and 10 mL of heat-inactivated rabbit serum.
6. Add the enriched EMJH supplement to the Medium Base
EMJH. Sterilize by filtration through a 0.22 μm filter system,
and create aliquots as needed for experiments. The final EMJH
medium can be stored at 4
C.
3.2 Establishing
Polarized Cell
Monolayers
1. Thaw a working aliquot of your eukaryotic cells into a T25 flask
with 5–7 mL of complete growth medium (see Note 3).
2. Incubate at 37
C with 5% CO 2 for growth until cells reach
approximately 80–100% confluence.
3. Wash cells twice with 3 mL of PBS.
4. Add 1 mL of trypsin/EDTA solution for trypsinization.
5. Incubate at 37
C with 5% CO 2 until cells are completely
disassociated.
6. Add 2 mL of complete growth medium and passage ~1.5 mL
of trypsinized cells into 10–15 mL of complete growth
medium in a T75 flask.
7. Incubate at 37
C with 5% CO 2 for growth.
8. Once the cells have reached an approximate 80–100% confluence, wash them twice with 9 mL of PBS.
9. Add 2 mL of trypsin/EDTA solution for trypsinization.
10. Incubate at 37
C with 5% CO 2 until cells are completely
disassociated.
11. Add 4–6 mL of complete growth medium and transfer the cells
into a 15 mL tube.
12. Enumerate the eukaryotic cells with a Neubauer counting
chamber (see Note 4).
13. Dilute the cells in complete growth medium to reach a concentration of 4 Â 10
5 cells/mL.
14. Add 1.5 mL of complete growth media to the lower compartment of each well on the plate (see Note 5).
15. Put the plate at 37
C with 5% CO 2 for 20 m to equilibrate.
164
Elsio A. Wunder Jr.
CaCl 2 , MgCl 2 , ZnSO 4 , and vitamin B12. Also add 10 mL of
FeSO 4 , 12.5 mL of Tween 80, and 0.1 mL of MgSO 4 stock
solutions. Adjust pH to 7.4 with NaOH and adjust volume to
100 mL with water. The EMJH supplement can be stored at
À20
C indefinitely until use.
4. For 1 L of EMJH medium, dissolve 2.3 g of Difco Leptospira
Medium Base EMJH in 890 mL of water. Add 0.9 mL of 10%
glycerol stock solution and adjust pH to 7.4 with HCl.
5. Thaw 100 mL of EMJH supplement and add 1 g of lactalbumin hydrolysate, 0.04 g of sodium pyruvate, 100 μL of SOD
stock solution, and 10 mL of heat-inactivated rabbit serum.
6. Add the enriched EMJH supplement to the Medium Base
EMJH. Sterilize by filtration through a 0.22 μm filter system,
and create aliquots as needed for experiments. The final EMJH
medium can be stored at 4
C.
3.2 Establishing
Polarized Cell
Monolayers
1. Thaw a working aliquot of your eukaryotic cells into a T25 flask
with 5–7 mL of complete growth medium (see Note 3).
2. Incubate at 37
C with 5% CO 2 for growth until cells reach
approximately 80–100% confluence.
3. Wash cells twice with 3 mL of PBS.
4. Add 1 mL of trypsin/EDTA solution for trypsinization.
5. Incubate at 37
C with 5% CO 2 until cells are completely
disassociated.
6. Add 2 mL of complete growth medium and passage ~1.5 mL
of trypsinized cells into 10–15 mL of complete growth
medium in a T75 flask.
7. Incubate at 37
C with 5% CO 2 for growth.
8. Once the cells have reached an approximate 80–100% confluence, wash them twice with 9 mL of PBS.
9. Add 2 mL of trypsin/EDTA solution for trypsinization.
10. Incubate at 37
C with 5% CO 2 until cells are completely
disassociated.
11. Add 4–6 mL of complete growth medium and transfer the cells
into a 15 mL tube.
12. Enumerate the eukaryotic cells with a Neubauer counting
chamber (see Note 4).
13. Dilute the cells in complete growth medium to reach a concentration of 4 Â 10
5 cells/mL.
14. Add 1.5 mL of complete growth media to the lower compartment of each well on the plate (see Note 5).
15. Put the plate at 37
C with 5% CO 2 for 20 m to equilibrate.
164
Elsio A. Wunder Jr.