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SF9 Cells

Sf9 insect cells are grown in suspension in serum-free medium. They are routinely maintained in flasks at densities between 3x105 and 7x106 cells/ml to keep logarithmic growth by passaging 2 times per week. To freeze cells for later use, they are expanded to high densities, mixed with freezing medium containing DMSO, then cooled slowly and stored in liquid nitrogen. Cell density and viability are assessed using a hemocytometer and trypan blue staining.

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216 views3 pages

SF9 Cells

Sf9 insect cells are grown in suspension in serum-free medium. They are routinely maintained in flasks at densities between 3x105 and 7x106 cells/ml to keep logarithmic growth by passaging 2 times per week. To freeze cells for later use, they are expanded to high densities, mixed with freezing medium containing DMSO, then cooled slowly and stored in liquid nitrogen. Cell density and viability are assessed using a hemocytometer and trypan blue staining.

Uploaded by

manojkumar008
Copyright
© Attribution Non-Commercial (BY-NC)
We take content rights seriously. If you suspect this is your content, claim it here.
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Sandra Marcus, March 1, 2004

Growth and Maintenance of SF9 Cells

Spodoptera frugiperda (Sf9) cells insect cells grow in suspension, in serum-free medium (SFM).
Sf-900 II SFM can be ordered through Biochemistry Stores. Invitrogen cat# 10902

Flasks:

Use the sterile, disposable 125 ml and 500 ml flasks from Corning. The 50 ml size is not
available from Corning.

For small culture volumes (10 ml), use Nalgene polycarbonate shaker flasks. Before use, rinse
several times in MQ water and let air dry. To autoclave: Set the cap on top without engaging
the threads. Cover with a piece of foil to secure the cap in place. Autoclave on a dry cycle, 15
min sterilize 121°C, 15 min dry. Discard after use.

Flask working volumes:

50 ml flask: 10 ml
125 ml flask: 20-50 ml
500 ml flask: 125-200 ml

Thawing cells

1. Remove a vial from liquid nitrogen and maintain on dry ice.


2. Prewarm some SFM.
3. Thaw the vial in a 37°C waterbath by gently agitating. The O-ring and cap should be kept
out of the water to prevent contamination. Remove the vial from the water as soon as the
contents are thawed. Wear safety goggles, since vial may explode.
4. After thawing, spray the entire vial with 70% EtOH
5. Using a 2 ml pipet, gently suspend any settled cells and transfer the vial contents to a
sterile 15 ml tube.
6. Slowly add 10 ml prewarmed SFM.
7. Pellet cells by centrifuging 1000 rpm for 5 min. Aspirate the media.
8. Gently flick the tube to dislodge the pellet.
9. Add 10 ml fresh media and incubate at 27°C 120 rpm. Cells should be 1-2× 106 cells/ml.
10. Check cell count and viability 48 hours later. If you want to expand cells for freezing,
proceed to "Expanding and Freezing SF9 cells". Otherwise, proceed to "Routine
maintenance of SF9 cultures".

Routine Maintenance of SF9 Cultures


Develop a maintenance schedule to keep the culture at an appropriate density, nutrient level and
pH.
1. Check cell density and viability (see below).
2. To maintain a culture volume of 10 ml: Draw up the entire volume of cells into a 10 ml
pipet. Return an appropriate volume into the same flask. Harvest the remainder for
experiment use, or discard them into the bleach bucket.

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Sandra Marcus, March 1, 2004

3. Add an appropriate volume of fresh SFM and place the flask back into the incubator.
4. Cells should be maintained at a density between 3× 105 and 7× 106 cells per ml to keep
them growing log rhythmically. This means you passage them about 2 times per week.
Use the following as a guide:
If you will leave them 4 nights, dilute them to 3× 105/ml.
If you will leave them 3 nights dilute them to 5 to 8× 105/ml.
If you will leave them 2 nights, dilute them to 1× 106 cell/ml.
5. Keep careful records of the cell passage number with dates, cell density and cell viability.
6. Once every 3 weeks, gently centrifuge the cell suspension at 100× g (800 rpm) for 5
min. Remove all the old medium. Gently flick the tube to dislodge the cells. Resuspend
the cells in the same volume of fresh SFM. Transfer to a new flask. This procedure will
reduce the accumulation of cell debris and metabolic byproducts which can slow cell
growth.

Expanding and Freezing SF9 cells

1. Cells must be growing well with good viability or they will not withstand freezing.
2. Gradually expand the cell culture by moving to a 125 ml and then a 500 ml flask, using
the volume guidelines above. Maintain cultures at the densities given above.
3. To freeze about 25 vials, grow a 100 ml culture in a 500 ml flask so the cells are in log
phase (about 4-6× 106 cell/ml).
4. Transfer to two sterile 50 ml centrifuge tubes, enough cells to give about 1.8× 108 total
cells per tube.
5. Centrifuge 1000 rpm for 5 min.
6. Withdraw 14 ml of the used media and transfer to a fresh tube. This "conditioned
medium" is used to make your freezing media.
7. Aspirate the remaining media from the cells and discard.
8. To the 14 ml conditioned medium, add: 14 ml fresh SFM
2.3 ml cell culture grade DMSO
Filter sterilize through a 0.2 µ m syringe filter (Millipore PVDF Durapore membrane).
Final proportions are: 50% conditioned medium/50% fresh medium/7.5% DMSO.
9. Flick each tube gently to dislodge the cells. Suspend each pellet in 12 ml freezing
medium by pipeting up and down gently. Final cell density should be between 1 and
2× 107 cell/ml.
10. Aliquot 1 ml each into sterile cryovials labeled with the following information:
SF9 cells and passage #
Date
Total number of cells
Lab book info
11. Make no more than 20-25 vials at once time.
12. After cells have been aliquoted, place them on ice.
13. Put them into an insulated foam box and into the -80°C freezer for a slow freeze.
14. After 24-72 hours, cells must be transferred to liquid nitrogen storage. See Sandra for
this.

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COUNTING CELLS
→ Mix 450µ l 0.1% Trypan Blue in PBS(dye) with 50 µ l cell suspension. Mix gently but
thoroughly.
→ Transfer 10 µ l of the above mixture to counting chamber (with cover glass on top)
→ Count number of cells within "counting squares"
→ Example calculation:
counts = 150, 140
average = 145
145/4 x 104 x 10 (dilution factor) = 3.62 x 106 cells/ml

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