High Five™ Cells (BTI-TN-5B1-4) are a clonal isolate derived from the parental Trichoplusia ni cell line (cabbage looper ovary) and are commonly used for the expression of recombinant proteins using the #Baculovirus Expression Vector System (BEVS).
Protocol:
Day -1: 1. Maintain High Five cells in #ESF921 media supplemented with 18mM L-Glutamine at 27C and 120 RPM with 25mm orbital shaking diameter.
2. Maintain #SF9 cells in Grace's Insect media supplemented with 6mM L-Glutamine.
Day 0: 1. Plate 0.8 million #SF9 cells or #SF21 cells in a well of a 6-well plate. Allow the cells to attach to the plate for 15 mins at room temperature inside Bio Safety Cabinet. Replace the media with plating medium (Grace's+6mM L-Glutamine+10% FBS).
2. Invert mix the #Cellfectin II reagent and dilute 8uL of it in 100uL of #Grace's and mix gently.
3. Dilute 1-3ug of #bacmid DNA into 100uL of Grace's and mix gently (DNA volume should not go beyond 10uL).
4. Add the diluted #DNA to diluted #Cellfectin II Reagent, mix gently and incubate at room temperature for 20-30 minutes.
5. Add the complex dropwise to the cells. Incubate the cells at 27C for 3-5 hours.
7. Incubate the cells at 27C for 72-96 hours or till you see signs of viral infection.
Day 4: 1. Collect the culture at high viability (~80%) and spin down at 1500 rpm for 15-20 mins at 4C. Add 2% FBS to the supernatant (P0 #virus stock) and store at 4C (short-term) or -80C (long-term), protected from light.
2. Titer the P0 virus stock by plaque assay/ #qPCR.
Day 5: 1. Seed 1 to 1.5 million #SF9 or #SF21 cells in an #Erlenmeyer flask with #ESF921 media supplemented with 6mM L-Glutamine. Allow the cells to accustom for 30 mins in shaker incubator at 120 RPM.
2. Use 0.1 MOI of P0 virus to infect the culture.
Inoculum required (mL)= (Desired MOI in pfu/cell X total no. of cells) / Titer in pfu/mL.
3. Incubate the cells at 27C for 48-72 hours and check for signs of viral infection.
Day 7: 1. Collect the culture at high viability (~80%) and spin down at 1500 rpm for 15-20 mins at 4C. Add 2% #FBS to the supernatant (P1 #virus stock) and store at 4C (short-term) or -80C (long-term), protected from light.
2. Titer the P1 virus stock by plaque assay/ #qPCR.
Day 8: 1. Seed 1.5 to 2 million High Five cells in an Erlenmeyer flask with #Express Five media supplemented with 18mM L-Glutamine. Allow the cells to accustom for 30 mins in shaker incubator at 120 RPM.
2. Use 0.5 to 1.5 MOI of P1 virus to infect the culture.
3. Incubate the cells at 27C for 48-72 hours and check for signs of viral infection.
Day 10: 1. Collect the culture at high viability (70-80%) and harvest by spinning at 4000-6000 RPM at 4C for 30 mins. Collect the supernatant for secretory protein and add 0.02-0.03% sodium azide, and store at 4C. For intracellular protein, spin the culture at 2000 RPM and wash the pellet with #PBS before proceeding to purification.
Concise protocol, thanks!