3. Add 10 mL DMEM and gently tap the sides of the culture
dishes to detach the cells. Pipette cells up and down using a
10 mL serological pipette, until all clumps are dispersed and a
homogenous suspension is obtained.
4. Transfer the cell suspension to Falcon tubes and centrifuge at
1200 Â g for 2 min. Remove the supernatant and resuspend the
pellet in 10 mL complete DMEM.
5. Transfer cell suspensions from 5 Â 55 cm
2 petri dishes or
4 Â 75 cm
2 flasks to 1700 cm
2 ribbed-surface roller bottles
and add 240 mL complete DMEM. Grow the cells in a Roll-In
CO 2 Control Incubator for 72 h.
6. Replace cell media with 200 mL fresh, complete DMEM.
Transfect the HEK293T cells either with pCi-S or pCi-S/
preS1
21–47 plasmids, using a mixture of 50 mL TB, 750 μL
TR, and 500 μg plasmid DNA per bottle (see Note 7). Keep a
bottle of nontransfected HEK293T cells as control. Return
cells to the Roll-In CO 2 Control Incubator for 72 h.
7. Collect cell media and clarify by centrifugation at 10,000 Â g,
for 10 min, at 4
C. Store at 4
C until further use.
8. Add 200 mL fresh, complete DMEM to the cells and return the
bottles to the incubator for another 72 h period (see Note 13).
9. Repeat step 3.
10. Harvest cells by flushing up and down 50 mL PBS using a
10 mL serological pipette. Centrifuge cell suspensions at
1200 Â g for 10 min. Weigh resulting pellets and store at
À20
C until further use.
11. Analyze expression and secretion of HBV antigens by ELISA
and western blot, as above.
3.4 Antigen
Purification
1. Pour 4 mL of 20% sucrose in 38.5 mL ultracentrifuge tubes.
Gently add 34 mL media on top of the sucrose cushion. Centrifuge samples at 32,000 rpm (125,755 Â g, SW32Ti rotor)
for 5 h, at 4
C.
2. Carefully remove supernatants by using a serological pipette,
leaving about 0.8 mL of samples at the bottom of the tube.
Add 0.2 mL PBS and leave the tubes overnight on ice (see Note
14).
3. Gently vortex the tube for 10 s and pool resuspended pellets
corresponding to a specific antigen to a final volume of 1.7 mL.
4. Analyze secretion of HBV antigens by ELISA and western blot,
as in Subheading 3.2. Include transfected HEK293T cell
lysates to compare migration patterns of intra- and extracellular
antigens (Fig. 3) (see Note 15).
5. Pipet 2 mL of each of 60%, 45%, 35%, 25%, and 15% sucrose
solutions in a 12 mL ultracentrifuge tube, starting with the
higher concentration at the bottom. Gently layer the 1.7 mL of
HBV Surface Antigens in Mammalian Cells
89
dishes to detach the cells. Pipette cells up and down using a
10 mL serological pipette, until all clumps are dispersed and a
homogenous suspension is obtained.
4. Transfer the cell suspension to Falcon tubes and centrifuge at
1200 Â g for 2 min. Remove the supernatant and resuspend the
pellet in 10 mL complete DMEM.
5. Transfer cell suspensions from 5 Â 55 cm
2 petri dishes or
4 Â 75 cm
2 flasks to 1700 cm
2 ribbed-surface roller bottles
and add 240 mL complete DMEM. Grow the cells in a Roll-In
CO 2 Control Incubator for 72 h.
6. Replace cell media with 200 mL fresh, complete DMEM.
Transfect the HEK293T cells either with pCi-S or pCi-S/
preS1
21–47 plasmids, using a mixture of 50 mL TB, 750 μL
TR, and 500 μg plasmid DNA per bottle (see Note 7). Keep a
bottle of nontransfected HEK293T cells as control. Return
cells to the Roll-In CO 2 Control Incubator for 72 h.
7. Collect cell media and clarify by centrifugation at 10,000 Â g,
for 10 min, at 4
C. Store at 4
C until further use.
8. Add 200 mL fresh, complete DMEM to the cells and return the
bottles to the incubator for another 72 h period (see Note 13).
9. Repeat step 3.
10. Harvest cells by flushing up and down 50 mL PBS using a
10 mL serological pipette. Centrifuge cell suspensions at
1200 Â g for 10 min. Weigh resulting pellets and store at
À20
C until further use.
11. Analyze expression and secretion of HBV antigens by ELISA
and western blot, as above.
3.4 Antigen
Purification
1. Pour 4 mL of 20% sucrose in 38.5 mL ultracentrifuge tubes.
Gently add 34 mL media on top of the sucrose cushion. Centrifuge samples at 32,000 rpm (125,755 Â g, SW32Ti rotor)
for 5 h, at 4
C.
2. Carefully remove supernatants by using a serological pipette,
leaving about 0.8 mL of samples at the bottom of the tube.
Add 0.2 mL PBS and leave the tubes overnight on ice (see Note
14).
3. Gently vortex the tube for 10 s and pool resuspended pellets
corresponding to a specific antigen to a final volume of 1.7 mL.
4. Analyze secretion of HBV antigens by ELISA and western blot,
as in Subheading 3.2. Include transfected HEK293T cell
lysates to compare migration patterns of intra- and extracellular
antigens (Fig. 3) (see Note 15).
5. Pipet 2 mL of each of 60%, 45%, 35%, 25%, and 15% sucrose
solutions in a 12 mL ultracentrifuge tube, starting with the
higher concentration at the bottom. Gently layer the 1.7 mL of
HBV Surface Antigens in Mammalian Cells
89