Hello Anastasiia Hubiernatorova,

The protocol seems okay. But there are some suggestions which you could incorporate and accordingly modify your protocol.

1. The pore size of the insert: It depends on the type of cells that are to be used in the assay. Choose the right pore size for your cells. Too large a pore size cannot be used for smaller cell types, but the pores can’t be so small either such that migratory cells cannot fit through even in the presence of the chemoattractant. A 5µm pore size should be appropriate for certain cancer cell lines like MDA-MB-231 cells. 

2. The chemoattractant concentration used: Optimization of the chemoattractant of interest is an important step, especially when there is no data available for your cell line and FBS which is used as the chemoattractant. In such case, it will be good if you run the assay with several different serial dilutions of the chemoattractant. Response of different cell types to the chemoattractant may vary. As far as I know for chemoattractant such as FBS, a concentration of 10% serum is a good start while you are optimizing your seeding density.

3. Qs. Is 130k of the cells enough (it's a 24-well plate transwell insert). Should be, but now I hesitate.

You may have to optimize the seeding density for MDA-MB-231. At a concentration that is too high, the cells could either be hard to count or they could oversaturate the pores in the membrane, while too few cells added could lead to inconsistent counting and results. A seeding density of 130k is too high. There is a possibility that the cells could have oversaturated the pores in the membrane. You should start with 25000 cells as the initial seeding density for a 24-well plate transwell insert during the optimization process.

4. Timing: The incubation time for the assay would also be important to optimize depending on the cell type and the chemoattractant that is used. Typically, 16-24 hours would be a good start, but some cell lines may take longer. Please make a note of this.

5. Qs. Maybe the concentration of Geltrex is too high and they just couldn't degrade it?

Do not dilute Geltrex by fold dilution. Dilute to a specific concentration. Every lot of Geltrex will have a specific protein concentration or dilution factor included in the Certificate of Analysis. The protein concentration varies between the lots and this specific protein concentration should be used to calculate the amount of Geltrex required for your application. For Matrigel, the coating solution is prepared by diluting Matrigel with chilled coating buffer to a final concentration of 200 µg/mL. For Geltrex, you need to optimize the final concentration. Gently swirl the diluted coating solution to mix and store on ice until further use. Use Chill pipette tips and other materials that come into contact with Geltrex. With the help of sterile forceps, place the insert into 24-well plate. Pipette 100uL of coating solution to the center of the apical side of the insert and without shaking, incubate the closed plate at 37°C for 2-3 hours. Once done, make sure to follow the next steps quickly to avoid the gel from drying out.

6. Qs. When I was discarding a medium (step 6) I used aspirator, could it be the cause? As far as I understand the method, invaded cells should have already attached to the bottom of the chamber, but if aspiration somehow could detach them?

Pick up the cell culture insert using forceps and then transfer the insert to a clean gloved hand to aid with grip when swabbing the insert. Ensure that you don’t touch the actual membrane itself in the process. Using a cotton-tipped applicator, gently remove any non-migrated cells from the apical side of the transwell insert membrane. Repeat to ensure that there are no more remaining non-migrated cells. Be very careful when removing the non-migratory/invasive cells from the apical side of the membrane. Too much pressure can lead to the membrane becoming detached or could displace some of the cells that have invaded.

Pipette 4% PFA into a well of a 24-well plate. Place the transwell insert into a well containing 4% PFA. Incubate at room temperature for 15-20 minutes to fix the migrated cells on the basal side of the transwell insert membrane. Wash twice with 1xPBS.

Place the transwell insert into the well containing 2% (w/v) crystal violet and incubate for 5-10 minutes at room temperature.

Remove the transwell insert and place it into a well containing PBS or water to wash any remaining crystal violet stain from the membrane. After washing, take images of the stained migrated cells under the microscope for qualitative analysis.

Include the above suggestions in your protocol and I hope you succeed.

Good Luck!

Dear Malcolm, thank you very much for your detailed reply!

I have already optimized the protocol, and the problem appeared to be Geltrex handling. In the protocol it's said that one should dilute it 1:4 before coating. However, after I coated the membrane, Geltrex appeared to be quite hard and it detached from the membrane when I pippeted the cells onto it, although I was careful. After this, I tried 2 different concentrations of Geltrex and 2 different concentrations of FBS, as well as added the uncoated control to ensure there's no problem with handling of the samples after incubation. Now, all the wells worked out with very nice results. Also I may note that indeed it's better not to use the aspirator and be very careful with the washing step prior to fixing the cells. Also, 130k cells per well seems just ok at least for my cells, but of course they may differ quite a lot from lab to lab.