I have a problems with Boyden chamber assay for HCT-116 cell lines, I did not see any stained cell in the membrane. I have had a success with other cell line, A549. Does anyone have any suggestion? Do I need to change something in the method? The lower chamber I put 32 uL DMEM with 10% FBS and FS, upper chamber: 51 uL DMEM and cells, I used matrigel-coating membrane and poretic polycarbonate 8uM membrane dish. After seeding in chamber, I incubated 6h in the incubator.
Thank you for your helping!
Hi
I worked with HCT116 cells for migration and invasion using boyden chamber. You need to use 500ul of DMEM (20% FBS) in bottom chamber and 50000 cells in the upper chamber in 300ul media (without FBS). Incubate for 12-24hours to observe the migration.
Also, you need to equillibriate the membrane with media (without FBS) for 1hr at least before seeding the cells.
Good luck
Anuj Anuj Thank you for advice! But there are something I am not clear in your protocol. I used 48 chamber, can I use 500 uL DMEM (20% FBS) in the bottom chamber, 300 uL media in the upper chamber. I coated the membrane with Matrigel, do I need to equilibriate the membrane with media. Thank you very much for your reply!
Hi Jenny,
Could you please clarify all the steps you have followed for this assay?
Adding to what Dr. Anuj has suggested- You will require fixing and staining cells after the migration/invasion period of 12-24 hours, to be able to properly visualize and count the migrated/invaded cells.
Good luck with your experiments.
Hi Jyoti Motwani!
Thank you for your advice. My Boyden chamber invasion assay is following these steps:
1. Seeding HCT-116 cell lines in 12-well plate (2x10^5 cell/well) in 24 hours.
2. Treat with drug in 24 h, coat the membrane with Matrigel (900 uL PBS+ 10uL Matrigel)
3. Seeding in chamber
Lower chamber: 31 uL medium + 10% FBS
Upper chamber: 52 uL medium without serum + cells (5000 cells/ chamber)
4. Staining
Fix the membrane in Methanol in 10 min
Stain with Liu stain A in 10 min, destain by rinsing in water (3 times)
Stain with Liu stain B in 10 min, destain by rinsing in water (3 times)
Drain the membrane.
Do you have any suggestion for me? I look forward your response!
Hi Jenny,
Please find my answers inline:
1. Seeding HCT-116 cell lines in 12-well plate (2x10^5 cell/well) in 24 hours.
2. Treat with drug in 24 h, coat the membrane with Matrigel (900 uL PBS+ 10uL Matrigel)
Its tricky to set the matrigel. It requires a lot of optimization before you get it right. I prefer using pre-coated matrigel inserts (Corning). You can refer to the document attached (TumourCellInvasionAssays.pdf) for more details on the concentration of matrigel.
3. Seeding in chamber Lower chamber: 31 uL medium + 10% FBS Upper chamber: 52 uL medium without serum + cells (5000 cells/ chamber)
The cell density and media volume is low. What plate did you use for inserts? You can refer to the document attached (an_Chemotaxis_protocol.pdf) for recommendations on cell density and media volume depending on the size of the well. However, it is always best to optimize the cell density before carrying out invasion experiments because the cell density may be different depending on the size of a cell. In your first post you mentioned that you carried out the invasion assay for 6 hours. It is possible that 6 hours is too less for cells to divide. I would suggest optimizing the invasion assay time of 6, 12, 24 and 48 hours and choose an invasion time depending on that. Some papers also show that FBS may not be a best chemoattractant for all cell types. But it would be best to try fixing the cell density, media volume and cell invasion first. If the assay still doesn't work with those changes, you may think of changing the chemoattractant.
4. Staining Fix the membrane in Methanol in 10 min
Before fixation :
i) Take out the media from inserts. Scrape the non-invaded cells from inside of the insert using a cotton swab dipped in media
ii) Wash the inserts first in 1X PBS before fixation. This would help in washing out media that would interfere with methanol fixation.
What temperature did you use for fixing the cells? It is recommended to fix the cells in -20C to avoid protein denaturation by 100% methanol. Wash the inserts with distilled water before staining.
Stain with Liu stain A in 10 min, destain by rinsing in water (3 times) Stain with Liu stain B in 10 min, destain by rinsing in water (3 times) Drain the membrane.
It would be best to dry the membrane overnight in an incubator at 37C prior to imaging and quantification.
Do you have any suggestion for me? I look forward your response!
Hope this helps.
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