Hello Jialei Hua

You may collect the conditioned medium by centrifuging for 10 min at 3,000 rpm at 4 degree C, and the supernatant may be 0.2 μm filtered to remove cell debris. Supernatant may be stored in aliquots at -80 deg C for further use. However, in selected experiments, you may concentrate the conditioned medium by ultrafiltration using Amicon Ultra-15 centrifugal filter devices with 10 kDa nominal molecular weight limit. For this purpose, 15 ml of conditioned medium may be collected in Amicon Ultra-15 tubes and centrifuged at 4000 g at 4 deg C for 25 mins.

Indirect co-cultures incorporate a physical separation between cell types, such as a semi-permeable membrane in the form of a transwell system, only enabling signaling via secretory factors.

You may use CAFs or their conditioned medium to induce cancer cell migration or invasion using indirect co-culture.

The steps for indirect co-culture for cell migration are as follows:

1. Add 500ul diluted conditioned media and monolayer cultured CAFs in growth media to a 24-well plate in their respective wells in triplicate.

2. Add 100ul of cancer cell suspension with an optimal seeding density onto the membrane of the 24-well transwell insert.

3. Use sterile forceps to transfer the transwell insert into each well of the 24-well plate already filled with diluted conditioned medium and the monolayered cultured CAFs (in growth media) in their respective wells.

4. Incubate the 24-well plate along with the transwell insert at 37°C and 5% CO2 for 24 hours.

5. Remove the plate from the incubator after 24 hours.

6. 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.

7. Pipette 1 mL of 70% ethanol into a well of a 24-well plate.

8. Place the transwell insert into a well containing 70% ethanol. Incubate at room temperature for 10–15 minutes to fix the migrated cells on the basal side of the transwell insert membrane.

9. Place the insert into an empty well to allow the transwell membrane to dry.

10. Place the transwell insert into the well with 0.2% (w/v) crystal violet and incubate for 3–5 minutes at room temperature.

11. Remove the transwell insert and place it into a well containing PBS or water to wash any remaining crystal violet stain from the membrane.

12. Repeat the wash with PBS or water to remove excess crystal violet.

13. Allow the membrane to dry by placing the transwell insert into an empty well.

14. Using a microscope, multiple images of the transwell membrane can be taken with a 10x or 20x objective to ensure a representative field of view is captured. Count the number of transmembrane cells. The mean value will represent the migratory cell number.

Similarly, you may use indirect co-culture for cell invasion assay. However, in cell invasion assay, the transwell insert membrane is coated with Matrigel. Incubate the transwell with Matrigel at 37°C for 30 min for gelling. Wash off the Matrigel of the downside of the membrane twice with pre-warmed serum-free culture medium. Then proceed with step 1 as in the above protocol.

Good Luck!

Cells are grown in a culture medium inside an incubator with controlled conditions of temperature, humidity, and CO2 levels. Once the cells have conditioned the medium (i.e., after they have grown and released substances into the medium), the medium is carefully aspirated using a pipette or an automated system to avoid disturbing the cell layer. The collected medium is then centrifuged at a low speed to remove any detached cells or debris. This step ensures that the conditioned medium is free from any cellular contamination.