Biology Reference
In-Depth Information
compared to various other cell lines that were tested. Reliable substitute cell lines that
can be used in order to express a desired protein of interest include HeLa or COS7
cells. In our hands, transfectability and FIT2 expression were superior in HEK293
cells. Alternatively, a high-throughput lipid-binding screen could be performed under
various conditions using an adenoviral or lentiviral expression system in other cell
lines if so desired; however, we have not explored these possibilities or set out to de-
fine optimal experimental conditions for such an approach.
11.2.1.2
Cell culture of insect cells
11.2.1.2.1
Spodoptera frugiperda Sf9 cell culture
Sf9 cells were cultured in TNM-FHmedium (
Trichopulsia ni
medium—Formulation
Hink), consisting of Supplemented Grace's Insect Cell Medium, supplemented
with the addition of 10% FBS-HI (American origin, Invitrogen), 1
penicillin-
streptomycin (Invitrogen), and 10
m
g/ml gentamicin (Sigma). Cells were initially
cultured in monolayers in tissue culture treated T75 flasks (Corning) until 90% con-
fluent (
10
7
cells) and passaged no more than 20 times. Cell viability was maintained
95% viable cells as determined by Trypan blue exclusion (1
m
l of 0.4% Trypan
blue in phosphate-buffered saline (PBS) with 10
m
l of cell suspension, measured on a
hemacytometer). Suspension cultures of
at
95% viable cells were generated by seeding
Sf9 cells into spinner flasks at 10
6
cells/ml at 27-28
C and at 80-90 rpmwithout CO
2
in the dark. Volume of media was kept at 30-50% of flask volume to optimize aeration
and shear stresses. Doubling time was approximately 48-72 h. Cells were passaged in a
sterile cell culture hood using large-volume electric pipettors when they reached a con-
centration of 2-2.5
10
6
cells/ml into fresh TNM-FHmedia by dilution. Cell densities
and viabilities were monitored daily throughout culture lifespan.
11.2.1.2.2
Trichopulsia ni High Five (Hi5) cell culture
High Five (Hi5) cells were grown in Express-Five SFMmedium, supplemented with
200 mM
L
-glutamine (GIBCO/Invitrogen) and 10
m
g/ml gentamicin in order to make
“Hi5 medium” at 27-28
C without CO
2
. Cells were initially seeded into T75 flasks
and grown as monolayers at similar densities to Sf9 cells, but never exceeding 90%
confluency. Cell densities, viabilities, and contamination were monitored daily and
maintained at
90-95%; otherwise, cells were discarded. Cells from monolayer
cultures were then seeded at a concentration of 10
6
cells/ml into suspension flasks
at 30-50% of flask volume in Hi5 medium supplemented with 10 U/ml heparin to
inhibit cell clumping. Hi5 cells were subsequently passaged each time they reached
a concentration of 2-2.5
10
6
cells/ml and weaned from heparin by dilution into
fresh Hi5 medium that did not contain heparin.
11.2.1.3
Baculovirus generation and amplification
In order to infect insect cells for large-scale protein production, baculovirus transfer
vectors were constructed using two different commercially available systems.
Using Novagen's BacMagic 2 system, cDNAs encoding N- or C-terminally
His
6
-StrepII-tagged recombinant proteins were amplified by PCR and products
