Generation of in vitro trained TAMs

Detailed Protocol:

A. Isolation of PBMC from buffy

Day 1:

1. Take 3 Leucosep tubes (227290, Sarstedt) for each buffy (around 50 mL blood).
2. Fill each tube with 15 mL of Ficoll (L6115, BIOCOLL Biochrom AG).
3. Centrifuge the tubes at 1600 rpm for 1 minute, at room temperature (RT).
4. Overlay approximately 15 mL of blood in each tube.
5. Carefully add PBS- 2mM EDTA up to 50 mL.
6. Centrifuge the tubes at 440 g for 30 minutes, at RT, and break set at 1.
7. Remove the upper part containing plasma.
8. Take PBMC layer from 3 tubes in a new 50 mL Falcon.
9. Fill up 50 mL Falcon with 1X RBC lysis buffer (555899, BD Biosciences) up to 50 mL
10. Incubate at RT for 15 minutes.
11. Centrifuge the tubes at 1600 rpm for 8 minutes, at room temperature (RT).
12. Carefully discard the supernatant.
13. Resuspend the pellet in 1 mL of PBS- 2mM EDTA  and fill the tube with PBS- 2mM EDTA  up to 50 mL.
14. Centrifuge the tubes at 1600 rpm for 8 minutes, at RT.
15. Repeat steps 12 to 15 for 2 more times.
16. Resuspend the pellet from 1 buffy in 432 mL of RPMI-1640 medium supplemented with 1% P/S (Note: 36 plates per buffy or dilute the PBMC according to the number of plates required).
17. Add 2 mL cell suspension/well in 6 well plates (83.3920.300, Sarstedt).
18. Incubate the plates in a 37^°C incubator for 2 hours.
19. After 2 hours, discard the medium and add 2mL/well macrophage medium (macro-medium - RPMI-1640 medium supplemented with 2% human serum and 1% P/S).
     

Day 2:

1. Discard the medium and add 2mL/well macrophage medium
    

Day 3:

1. Discard the medium and add 2mL/well macrophage medium

Day 5:

1. Discard the medium and add 2mL/well macrophage medium

Day 7:

1. Discard the medium and add 2mL/well macrophage medium

Day 9:

1. Around day 9, all unattached cells are washed out, and adherent macrophages are ready for the in vitro TAMs generation.

Notes:

• If the medium is not clear, then repeat step 23 every alternate day.
• With this protocol, approximate cell density is 1 x 10⁵ macrophages/well.
• Maintain the macrophages in culture for maximum 4 weeks with medium change on every alternateday.

B. Generation of in-vitro-trained TAMs

Day 1:

1. Harvest A549 cells with trypsin-EDTA, wash once with A549 cell medium (supplemented with 10% FCS and 1% P/S) and subsequently resuspend in macrophage medium.
2. Prepare A549 cell suspension – 1 × 10⁵ A549 cells/ 2mL /well in macrophage medium.
3. To generate M1-like TAMs, culture for 3 days.

Day 3: Part 1: Collection of M0 and M1-like TAMs

1. Collect the supernatant from M0 and 3-days co-culture (M1-like TAMs) for further experiments
2. Wash the cells with 2 mL 1X PBS once.
3. Collect M0 macrophages in PBS with a cell scraper, pellet down and store in -80 until further experiments.
4. To harvest M1-like TAMs, add 500µL/well trypsin-EDTA, incubate for 3 minutes in an incubator. (Note: Optimize trypsinisation time as per cancer cell line used for the co-culture. Trypsinisation time of more than 5 minutes affect the phenotype of macrophages)
5. Add 2 mL of macrophage medium to stop the reaction.
6. Shake the plate vigorously to detach cancer cells, discard the supernatant.
7. Repeat steps 32 and 33 twice with 1X PBS.
8. Collect M1-like TAMs macrophages in PBS with a cell scraper, pellet down and store in -80°C until further experiments.
    

Day 3: Part 2: Addition of new cancer cells to obtain M2-like TAMs on Day 5

1. Repeat steps 29 to 33 to detach cancer cells from plates
2. Wash the cells twice with a macrophage medium
3. Incubate in 37°C incubator for minimum1 hour in macrophage mediumbefore addition of new cells.
4. Repeat steps 25 and 26 to prepare A549 cell suspension.
5. Discard the medium from the plates from step 38, add new cells, and incubate for 2 days.

Day 5: Collection of M2-like TAMs

1. Repeat steps 28 to 35 to collect M2-like TAMs.

Notes:

• Some cancer cell lines undergo apoptosis in macrophage medium. Therefore, before starting co- culture with different cancer cell lines, first culture the cancer cells in a macrophage medium and compare its cell viability with its respective culture medium.
• This protocol is for 1 well of a 6-well plate. Scale up the volumes and cell numbers accordingly.
• Cancer cell lines, which need more than 5 minutes of trypsinisation for detachment, are not suitable for this co-culture, affecting the phenotype of macrophages.
   

Figure: Stepwise protocol for in vitro TAMs generation