2.2 Zika VLP
Expression
1. 2.5 M calcium chloride. Weigh 36.755 g of calcium chloride
(CaCl 2 ∙ 2H 2 O) into a glass beaker and dissolve in 80 mL of
deionized water using a magnetic stirrer. After the CaCl 2 has
completely dissolved, adjusted to a final volume of 100 mL
with deionized water. Filter the solution through a 0.22 μm
PES membrane filter and aliquot into 0.65 mL tubes for storage at 4
C.
2. 2Â HBS: 274 mM sodium chloride, 10 mM potassium chloride, 1.4 mM disodium hydrogen phosphate, 42 mM HEPES,
11 mM glucose. Weigh 8 g of sodium chloride (NaCl), 0.38 g
of potassium chloride (KCl), 0.1 g of disodium hydrogen
phosphate (Na 2 HPO 4 ), 5 g of HEPES and 1 g of glucose
(C 6 H 12 O 6 ) into a glass beaker and dissolve with 400 mL of
deionized water using a magnetic stirrer. After all the chemicals
have completely dissolved, adjusted the final volume to 500 mL
with deionized water using a measuring cylinder. Filter the
solution through a 0.22 μm PES membrane filter and aliquot
into 1.5 mL tubes for storage at 4
C.
3. Opti-MEM
® media (see Note 7). Pour 13.6 g of Opti-MEM
®
media powder into a beaker and dissolve it with 600 mL deionized water using a magnetic stirrer. In parallel, weigh 2.4 g of
sodium hydrogen carbonate (NaHCO 3 ) into a glass beaker and
dissolve it with 100 mL of deionized water using a magnetic
stirrer. After both solutions have dissolved completely, mix the
sodium hydrogen carbonate solution with the Opti-MEM
®
media. Adjust the final volume to 1 L with deionized water.
Filter the solution through a 0.22 μm PES membrane filter and
aliquot into 500 mL bottles and store at 4
C.
4. 1Â Dulbecco’s Modified Eagle’s Medium (DMEM). Pour
13.5 g of DMEM media powder into a beaker and dissolve it
with 600 mL deionized water using a magnetic stirrer. In
parallel, weigh 3.7 g of sodium hydrogen carbonate
(NaHCO 3 ) into a glass beaker and dissolve it with 100 mL of
deionized water using a magnetic stirrer. After both solutions
have dissolved completely, mix the sodium hydrogen carbonate
solution with the DMEM media. Adjust pH to 7.2 with concentrated hydrochloric acid (HCl) (see Note 8). Adjust the final
volume to 1 L with deionized water. Filter the solution
through a 0.22 μm PES membrane filter and aliquot into
500 mL bottles and store at 4
C.
5. HEK293T/17 cells cultured in 1Â DMEM supplemented
with 10% fetal bovine serum in an incubator with 5% CO 2 at
37
C.
6. 0.22 μm nitrocellulose membrane.
Zika Virus-Like Particles
187
Expression
1. 2.5 M calcium chloride. Weigh 36.755 g of calcium chloride
(CaCl 2 ∙ 2H 2 O) into a glass beaker and dissolve in 80 mL of
deionized water using a magnetic stirrer. After the CaCl 2 has
completely dissolved, adjusted to a final volume of 100 mL
with deionized water. Filter the solution through a 0.22 μm
PES membrane filter and aliquot into 0.65 mL tubes for storage at 4
C.
2. 2Â HBS: 274 mM sodium chloride, 10 mM potassium chloride, 1.4 mM disodium hydrogen phosphate, 42 mM HEPES,
11 mM glucose. Weigh 8 g of sodium chloride (NaCl), 0.38 g
of potassium chloride (KCl), 0.1 g of disodium hydrogen
phosphate (Na 2 HPO 4 ), 5 g of HEPES and 1 g of glucose
(C 6 H 12 O 6 ) into a glass beaker and dissolve with 400 mL of
deionized water using a magnetic stirrer. After all the chemicals
have completely dissolved, adjusted the final volume to 500 mL
with deionized water using a measuring cylinder. Filter the
solution through a 0.22 μm PES membrane filter and aliquot
into 1.5 mL tubes for storage at 4
C.
3. Opti-MEM
® media (see Note 7). Pour 13.6 g of Opti-MEM
®
media powder into a beaker and dissolve it with 600 mL deionized water using a magnetic stirrer. In parallel, weigh 2.4 g of
sodium hydrogen carbonate (NaHCO 3 ) into a glass beaker and
dissolve it with 100 mL of deionized water using a magnetic
stirrer. After both solutions have dissolved completely, mix the
sodium hydrogen carbonate solution with the Opti-MEM
®
media. Adjust the final volume to 1 L with deionized water.
Filter the solution through a 0.22 μm PES membrane filter and
aliquot into 500 mL bottles and store at 4
C.
4. 1Â Dulbecco’s Modified Eagle’s Medium (DMEM). Pour
13.5 g of DMEM media powder into a beaker and dissolve it
with 600 mL deionized water using a magnetic stirrer. In
parallel, weigh 3.7 g of sodium hydrogen carbonate
(NaHCO 3 ) into a glass beaker and dissolve it with 100 mL of
deionized water using a magnetic stirrer. After both solutions
have dissolved completely, mix the sodium hydrogen carbonate
solution with the DMEM media. Adjust pH to 7.2 with concentrated hydrochloric acid (HCl) (see Note 8). Adjust the final
volume to 1 L with deionized water. Filter the solution
through a 0.22 μm PES membrane filter and aliquot into
500 mL bottles and store at 4
C.
5. HEK293T/17 cells cultured in 1Â DMEM supplemented
with 10% fetal bovine serum in an incubator with 5% CO 2 at
37
C.
6. 0.22 μm nitrocellulose membrane.
Zika Virus-Like Particles
187