ExpiSf Expression System Protocol Guide

Learn the fundamentals of protein expression using the Gibco ExpiSf Expression System with detailed overviews of the unique system components and key workflow steps for which optimal techniques will help maximize results. This protocol guide includes required material checklists, visual step-by-step guides, and helpful tips.

 

At the end of this protocol guide, you should be able to:

  • Outline the full ExpiSf Expression System workflow, identify key components of the system, and know when, how, and why to use each one
  • Apply optimal techniques for thawing and subculturing Gibco ExpiSf9 Cells to achieve maximum viability and cell health
  • Apply optimal techniques freezing, transfecting and infecting Gibco ExpiSf9 Cells to achieve maximum viability and cell health (available only in the Protein Expression Learning Labs)
  • Demonstrate best practices for efficient protein harvest as well as accurate detection and quantitation of protein yield (available only in the Protein Expression Learning Labs)
  • Identify workflow steps for which optimization will have the most impact to help improve future expression results (available only in the Protein Expression Learning Labs)

Discover the Protein Expression Learning Labs

Experience the ExpiSf expression system interactive protocol in an immersive and interactive lab environment.

Overview and components of ExpiSf Expression System Starter Kit

ExpiSf Expression System overview

The ExpiSf Expression System is the first chemically defined baculovirus expression system for high-yielding protein expression in Sf9 insect cells. Undefined media components like hydrolysates can make it quite difficult to get consistent results with Sf9 cells. The ExpiSf Expression System has been optimized to work with chemically defined Gibco ExpiSf CD Medium, which is free of hydrolysates, serum, protein, and animal-origin components. Transfection, baculovirus production, and recombinant protein expression all take place directly in ExpiSf CD Medium, providing consistency from start to finish.

 

ExpiSf Expression System Starter Kits provide enough cells, culture medium, and reagents to infect 1 L of cell culture. The kits also include the Gibco Bac-to-Bac Baculovirus Expression System for generation of bacmid DNA. With the exception of Gibco MAX Efficiency DH10Bac Competent Cells, all components of ExpiSf Expression System Starter Kits are animal origin–free (AOF).

Properties of ExpiSf9 Cells

  • Derived from the same parental lineage as the Gibco Sf9 cell line
  • Recover rapidly after thawing
  • Adapted to high-density suspension growth in ExpiSf CD Medium
  • Doubling time of approximately 24 hours during log phase growth
  • Broad log phase growth window of 4 x 106 to 12 x 106 cells per mL
  • Maximum cell density of approximately 2 x 107 cells per mL in shake flask culture
  • Stable growth and expression over multiple passages
  • High-yielding baculovirus production and protein expression

Components of ExpiSf Expression System Starter Kit (Cat. No. A38841)

 

Component

Quantity

Storage

ExpiSf9 Cells (1 x 107 cells/mL)

2 x 1.5 mL

Liquid nitrogen, vapor phase*

ExpiSf CD Medium

1 L

2°C to 8°C (protect from light)

ExpiSf Enhancer

4 x 1 mL

2°C to 8°C (protect from light)

ExpiFectamine Sf Transfection Reagent

1 mL

2°C to 8°C (do not freeze)

Opti-MEM I Reduced Serum Medium

500 mL

2°C to 8°C (protect from light)

pFastBac 1 Expression Vector (0.5 µg/µL)**

10 µg

–20°C

pFastBac 1-Gus Control Vector (0.2 ng/µL)**

4 ng

–20°C

MAX Efficiency DH10Bac Competent Cells

 

5 x 100 µL

Competent cells: –80°C

pUC19 DNA: –20°C

SOC Medium: 4°C or room temperature

Nalgene Single-Use PETG 125 mL Erlenmeyer Flasks (vented, non-baffled)

24

Room temperature

* Store the frozen cells in liquid nitrogen until ready to use. Do not store the cells at −80°C.

** In TE buffer, pH 8.0: 10 mM Tris-HCl, 1 mM EDTA, pH 8.0.

Protein expression essentials checklist

Protein expression essentials checklist

Recommended consumables, instruments, and equipment for protein expression with the ExpiSf Expression System.

Key laboratory instruments and equipment

Key laboratory consumables

Biosafety cabinet with HEPA filter

Incubator with temperature, humidity, and CO2 regulators

Orbital shaker

Water or metal bead bath

Centrifuge (>3,000 x g)

Vortex mixer

Refrigerator and freezer

Reagents and equipment for determining viable cell density and percent viability (automated cell counter, trypan blue solution)

Liquid nitrogen freezer

Aspiration pump (peristaltic or vacuum)

Waste containers and vacuum traps

Inverted microscope

Cell culture flasks, vented, non-baffled (125 mL to 5 L)

Serological pipettes and pipettors

(1 mL to 50 mL)

Multichannel pipettors and pipettes (1 µL to 200 µL)

Single channel pipettors and pipettes (1 µL to 1 mL)

Conical tubes

Cryovials

Permanent markers and labels

DMSO

Cell culture medium, PBS, and sterile H2O

70% ethanol

10% bleach

Personal protective equipment (PPE) (laboratory coat, safety goggles, disposable gloves, eye wash station)

Guidelines for growing and maintaining ExpiSf9 cells

General cell handling
  • All solutions and equipment that come into contact with cells must be sterile. Always use proper sterile technique and work in a laminar flow hood.
  • ExpiSf9 Cells are supplied in a vial containing 1.5 mL of cells at 1 × 107 viable cells per mL in 92.5% ExpiSf CD Medium and 7.5% DMSO.
  • Avoid exposing the cells to extreme temperature changes, even brief ones. When storing cells in liquid nitrogen following receipt on dry ice, allow the cells to remain in liquid nitrogen for 3–4 days before thawing.
  • On receipt, grow and freeze multiple vials of ExpiSf9 Cells to ensure you have a sufficient supply of early passage cells.
  • Transfer cells into ExpiSf CD Medium that has been warmed to room temperature when thawing or subculturing.
  • Always mix cells with gentle swirling. Avoid vigorous shaking or pipetting.
  • Ensure the cells are established by allowing freshly thawed cells to recover in culture for two or more passages before transfection or infection.
  • Passage cells when they reach a density of 5 x 106 to10 x 106 viable cells per mL during early or middle log phase growth, typically every 3–4 days.

Note: Do not subculture cells until they reach a density of at least 5 x 106 viable cells per mL. Cells that are subcultured at lower densities can have longer doubling times and produce lower titers over time. Modify the seeding density to attain the target cell density of at least 5 x 106 viable cells per mL at the subculturing stage.
  • Use a hemocytometer and the trypan blue exclusion method or an automated cell counter to determine cell viability. Make sure log phase cultures are ≥90% viable.

Medium
  • For suspension growth and protein expression, use ExpiSf CD Medium without any supplementation.

Important: ExpiSf CD Medium is light-sensitive. For optimal results, protect the medium from light during use and storage.

Thawing and establishing ExpiSf9 cells

Required materials

Step-by-step protocol for thawing and establishing ExpiSf9 Cells

 

Thaw cells (Day 1)

1. Remove the vial of cells from liquid nitrogen and swirl it in a 37°C water bath for 1–2 minutes to thaw the cells until only a small amount of ice remains. Do not submerge the vial in the water bath.

2. Just before the cells are completely thawed, decontaminate the exterior surface of the vial by wiping it down with 70% ethanol. When thawing is complete, open the vial in a laminar flow hood.

Add cells to medium (Day 1)

    

3. Use a 2 mL or 5 mL pipette to transfer the entire contents of the vial into the sterile 125 mL Erlenmeyer shake flask containing 25 mL of ExpiSf CD Medium warmed to room temperature.

Incubate cells

 

4. Incubate the cells on the orbital shaker at 27°C in the non-humidified, air-regulated incubator.

Note: Set the shaking speed to 125 ± 5 rpm for shakers with a 19 mm or 25 mm shaking diameter or 95 ± 5 rpm for shakers with a 50 mm shaking diameter.

Count cells and determine viability (Day 3–5)

 

 

5. Three days post-thaw, determine the viable cell density and percent viability. Cell viability should be ≥80% by Day 3 post-thaw.

6. Continue to monitor cell density and viability. Subculture the cells once the culture has reached 5 x 106 to 10 x 106 viable cells per mL, typically 4–5 days post-thaw.

Subculturing ExpiSf9 cells

Before you begin

  • Because ExpiSf9 Cells are adapted to high-density cell culture, we recommend allowing them to reach a density of 5 x 106 to 10 x 106 viable cells per mL before subculturing.
  • You may observe some clumping during routine culture maintenance when the cells reach higher densities. This will not impact the performance of the cells during transfection or infection.
  • Use the viable cell density to calculate the volume of cell suspension required to seed a new shake flask according to the recommended seeding densities in Table 1 and the recommended culture volumes in Table 2.

Required materials

Table 1. Recommended seeding densities for routine maintenance of ExpiSf9 Cells.

Subculture timing

Recommended seeding density

For cells ready 3 days post-subculture

0.7 x 106 to 1.0 x 106 viable cells per mL

For cells ready 4 days post-subculture

0.4 x 106 to 0.6 x 106 viable cells per mL

Table 2. Recommended volumes and shaking speeds for routine maintenance of ExpiSf9 Cells.

Flask size

Culture volume

Shaking speed*

125 mL

25–30 mL

19 mm shaking diameter: 125 ± 5 rpm

25 mm shaking diameter: 125 ± 5 rpm

50 mm shaking diameter: 95 ± 5 rpm

250 mL

50–60 mL

500 mL

100–120 mL

1 L

200–240 mL

2 L

400–480 mL

3 L

600–800 mL

80 ± 5 rpm (19 mm shaking diameter)

* Use upper ranges of recommended shaking speeds for larger culture volumes.

Step-by-step protocol for subculturing ExpiSf9 Cells

 

Passage cells (Day 1)

 

  

1. Use the viable cell density to calculate the volume of cell suspension required to seed a new shake flask according to the recommended seeding densities in Table 1 and the recommended culture volumes in Table 2.

2. Transfer the calculated volume of cells to fresh ExpiSf CD Medium warmed to room temperature in a shake flask.

Subculture cells

 

3. Incubate the cells on an orbital shaker in the 27°C non-humidified incubator until they reach a density of 5 x 106 to 10 x 106 viable cells per mL.

Note: Cells that are subcultured at densities outside this log phase growth window may have longer doubling times and produce lower protein titers over time. Modify the initial seeding density to attain the target cell density of 5 x 106 to 10 x 106 viable cells per mL at the subculturing stage.

4. Repeat Steps 1–3 to maintain or expand the cells for transfection or infection.

 

Note: If you want to freeze ExpiSf9 Cells, harvest the cells when they reach a viable cell density of 3 x 106 to 4.5 x 106 viable cells per mL with ≥90% viability. This will typically be on Day 3 after passaging cells from a seeding density of 0.5 x 106 to 0.6 x 106 cells/mL.

If the viable cell density is too low at the time of harvest, return the cells to the incubator for an extra day. If the viable cell density is too high at the time of harvest, subculture the cells at 0.5 x 106 to 0.6 x 106 cells/mL and prepare for harvest again after 3–4 days.

For Research Use Only. Not for use in diagnostic procedures.