2.6.1 Generation o f recom binant baculovirus clones
R e c o m b in a n t b a c u lo v iru s e s w e re p ro d u c e d by h o m o lo g o u s recom bination follow ing co-transfection o f 2 x 10^ Sf9 cells on 60 m m tissu e cu ltu re p la te s (N u n clo n ) w ith tra n s fe r v e cto r and B aculoG old DNA. The cells were seeded onto the plates, allow ed to a tta c h firm ly and the old m edium w as rem o v ed . In the c o tran sfectio n p late the m edium was rep laced by 1 m l o f G race's m edium with 10% FCS and to this a m ixture o f transfection buffer B, 5 pg recom binant baculovirus tran sfer vector D N A and 0.5 p g B aculoG old D N A was added in a dropw ise m anner w hile gently ro ck in g the plate. On the positive co n tro l p late the old m edium w as rep laced w ith 3 m l of fresh T M N -F H m edium . 50 p i o f w ildtype A cN PV baculovirus supernatant was added to this p late. On the negative control plate the old m edium was only replaced by 3 ml of fresh TM N -FH medium. A fter 4 hours the m edium was rem o v ed from the co -tra n sfec tio n p late, the p late was w ash ed
w ith T M N -FH m edium and then in cu b ated w ith fresh T M N -F H m edium at 27®C for 5 days. On the fifth day the supernatant was collected from the co transfection plate and checked, fo r expression of the heterologous protein.
2.6.2 Generation o f viral stocks
V iral stocks w ere p ro d u ce d by in fe c tio n o f Sf9 cells at low m ultiplicity. 2 x 10^ cells w ere seeded on 175 cm^ tissue cu ltu re flask s and in fected w ith 300 p i o f low tite r v iral stock. T he in fec te d cells w ere in cu b a te d fo r three days at 27® C and the su p ern atan t was h arv ested from the p lates on the th ird day by spinning the cells at 2,500 rpm in a H eraeus M inifuge T for 5 m in. The viral stocks were stored at 4®C in the dark.
2.6.3 Plaque assays
0.7 X 1 0^ Sf9 cells in log phase were seeded on each w ell o f a 6-
w ell-plate. The cells w ere allow ed to attach to the p late and w ere then infected with 1 m l o f serial dilutions o f the v iral stock (neat,
10'^, 10-4, 10-6, 1 0-8,) in p ro te in -fre e m edium . O ne w ell w as
tre a te d w ith p ro te in -fre e m edium w ith o u t v iru s as a n e g a tiv e control. The cells w ere incubated for 1 hour at 27®C. A sterile solution o f 3% low -m elting point agarose (Pharm ingen) in d istilled w ater was prepared, solubilised by heating in the m icrow ave and e q u ilib ra ted to 40®C. The solution was diluted 1:3 w ith com plete G race's m edium eq u ilib rated to 37®C. The p lates w ere asp irated and 3 m l o f the agarose solution w as added to each w ell and allow ed to set. The plates were then incubated for 6 days at 27®C.
The plaque titre was calculated using the formula:
plaque form ing unit (pfu)/m l = 1/d ilu tio n x n u m ber o f p laq u es x 1/ml inoculum.
2.6.4 Metabolic labeling o f insect cells
Ix 10^ in sect cells w ere seded onto 30 m m d ish es and in fected w ith the virus of in te re s t at an M O I o f 20. T he c ells w ere incubated for 70 hours at 27°C. Then the cells w ere starved fo r 1 h o u r in m eth io n in e-free, cy stein e-fre e G ra c e ’s m edium . F or the lab elin g each sam ple w as in cu b ated w ith 0.5 m l o f m eth io n in - free, cysteine-free G ra ce ’s containing 25 p C i o f Pro-M ix fo r one hour. A fter that time the cells were w ashed 3 tim es w ith PBS and in c u b a te d fo r a n o th e r h o u r w ith 0.5 m l PB S p e r sa m p le . Supernatant and cells were then harvested.
2.6.5 X-gal staining o f cells transfected with lacZ
The transfected cells w ere w ashed w ith PBS and then incubated in fix ativ e solution diluted 1:3 w ith PBS fo r three m ins. T he fix ed cells were w ashed with PBS, 3 ml o f X -gal staining solution was added and the cells were incubated at 37°C for 1 to 16 hours.
2.6.6 FACS staining
1 0 6 cells p e r sam p le w ere sta in e d w ith 1 p g /m l o f th e
appropriate m onoclonal antibody in a total volum e o f 100 pi. For internal staining of Raji cells the cells w ere fixed for 1 hour on ice w ith 0.6% form aldehyde and p erm eab ilized fo r 15 m in at 37°C w ith 0.2 % T w een-20 in PBS b efo re in c u b a tio n w ith the firs t antibody. A fter 1 hour in cu b atio n the cells w ere w ashed tw ice w ith PBS, 1% FCS, 0.002% sodium azide, in cu b ated for 30 m ins
w ith F IT C -c o n ju g a te d rab b it-an ti-m o u se P (a b ’)2-fra g m e n ts (1 :4 0
dilution) and washed again.
Flow cytom etric analysis was perform ed using a FA C Scan m achine (Becton Dickinson).
2.6.7 Transfection o f mammalian cells with lipofectamine
293 cells we^re grown to 50% co n flu en ce on 6 -w ell p lates and w ashed w ith O ptim em tran sfectio n m edium (G ibco). P er sam p le the follow ing com ponents were m ixed and incubated fo r 30 m ins: 100 jil Optim em containing the DNA (0.5 pg o f each plasm id) plus 100 p i O ptim em containing lipofectam ine (2 pi per 0.5 p g DNA). A fte r the in cu b atio n tim e an a d d itio n al 800 p i O ptim em w as added to each sam ple and the solution was carefully applied onto the cells. A fter 6 hours incubation at 37°C the cells w ere w ashed w ith DM EM , 10% FCS and kept in DM EM until use (usually 36-48 hours after transfection).
2.6.8 Electroporation o f mammalian cells
P e r sam p le l-2 x 10^ T2 cells w ere w ashed w ith serum - an d an tib io tics-free RPM I and resuspended at a d en sity o f 2-4x 10^ c ells/m l. To each sam ple 10 pi ste rile sh eared salm o n sp erm carrier DNA (10 mg/ml, Sigma) and 25 pg of each plasm id (sterile) w ere added. The sam ples w ere in cu b ated fo r 5 m ins at ro o m tem p era tu re . E lectro p o ratio n s o f ex actly 500 pi o f each sam p le w ere p erfo rm ed with the follow ing settings: 500 p F , 270 volts. A fter the electroporation the sam ples w ere left for 10 m ins and th en resu sp en d ed in 25 m l RPM I m edium c o n ta in in g FCS and antibiotics.