hMDMs differentiation and OR6A2 silencing protocol

02/2022, M. Orecchioni

Human Monocytes derived Macrophages (hMDMs) differentiation procedure:

Reagents

• EDTA-venous human blood (at least 100mL)
• PBS
• Ficoll-Paque (GE Healthcare)
• EasySep Human Monocyte Isolation Kit (StemCell Technologies, Cat # 19359)
• Complete RPMI media (10% FBS, +1% P/S) cRPMI
• rhM-CSF (100 µg/ml cat#300-25, Peprotech)
• 50 mL conical tubes
• Serological pipettes
• 6-well cell culture plates

Day 1

1. Peripheral blood mononuclear cells (PBMCs) isolation from EDTA-venous blood of healthy donors (25-50 years old) using a Ficoll-Paque (GE Healthcare) standard separation protocol.
2. Human monocytes enrichment from PBMCs by the EasySep™ Human Monocyte Isolation Kit (StemCell) according to the manufacturer’s instructions.
3. Resuspend enriched monocytes at 1/2×10⁶ cells/mL with 3mL culturemedium supplemented with 50 ng/ml rhM-CSF at 37 °C and 5% CO₂.
    

Day 4

4. Prepare 2 mL/wellcRPMI + 50 ng/mL M-CSF.Distribute 2 mL to each well, finalvolume in wells = 5 mL cRPMI + 50 ng/mL rhM-CSF.

Note: There will be a number of monocytes that are not attached, not all of the monocytes settle down onto the plate.

5. Incubate for 3 more days.

Day 7: siRNA knockdown of OR6A2 in hMDMs

Reagents

• Lonza Cell Line Nucleofector Kit V, Cat# VCA-1003
• Complete RPMI media (10% FBS, P/S)
• Silencer pre-designed OR6A2 siRNA (Thermo Fisher, #AM 16708)
• Silencer Negative Control No. 1 siRNA (Thermo Fisher, #AM4611)
• PBS (4°C)
• 0.5 M EDTA solution (4°C)
• Cell Stripper
• Sterile RNase-free water
• rhM-CSF (100 µg/ml cat#300-25, Peprotech)
• 50/15 mL conical tubes
• 1.5mL tubes

Detaching macrophages from plate:

6. Using 4°C PBS and EDTA, make a 2 mM EDTA solution in PBS. Use immediately or keep on ice until ready to use (50 mL volume).

7. Aspirate media and wash cells with 2 mL cold PBS.

8. Aspirate wash, and add 2 mL cold 2 mM EDTA-PBS solutionto each well.Incubate plate for 5-10 minutes at 37°C.

9. Pipet up and down in wells to detach cells using P1000 pipetter.

Note: Can use a cell scraper if the cells are not detaching. However, it is not recommended.

10. Transfer cells to conical tube.

11. Centrifuge cells 500 x g for 5 min, 4°C. Aspirate supernatant, resuspend cells in 1 mL 2 mM EDTA- PBS, count cells.

Electroporation and cell plating:

12. Determine number of cuvettes to use, and make appropriate volume of Nucleofector reagent, using about 2 million cells per cuvette (final volume, 100µL).

13. Prepared Nucleofector reagent by mixing 82 µL NF solution + 18 µL Solution 1 per cuvette. (4.5:1 Nucleofector solution: supplement)

14. Determine volume of cell suspension required for experiment, and transfer volume to 15 mL conical tube.

15. Spin down cells 500 x g, 5 minutes, aspirate PBS-EDTA (as much as possible without taking cells).

16. Resuspend pellets in Nucleofector reagent. Transfer volumes to 1.5mL tubes (e.g., 4 x10⁶ cellsin 200 uL Nucleofector reagent, to be split between 2 cuvettes).

17. Prepare siRNA dilution: Dilute 50 µM stock 1:10 to get 5 µM working stock with RNase free sterile dH₂O.

18. Add 1 µL/cuvette diluted siRNA (divide samples for SiRNA-CTRL and SiRNA-OR6A2 respectively) to 1.5mL tubes (final concentration is 50 nM, or 5 pmol siRNA per cuvette); pipet to mix.

NOTE: Aliquot diluted siRNA, store at -20°C and use 2-3 times, with 1 or 2 freeze/thaw cycles per 5 µM aliquots.

19. Pipet up and down carefully with P100 pipet, then transfer volume from 1.5mL tubes to cuvette.

Avoid bubbles.

20. Electroporate cells using Nucleofector 2b Device (Lonza) using the preset BMDM program. Complete 1 cycle per cuvette. Complete 3 or 4 cuvettes, add 1 mL cRPMI + 50 ng/mL M-CSF to cuvettes, then do another set of 3-4 cuvettes adding 1 mL cRPMI after electroporation as previous set. Repeat with remaining cuvettes

   a. Transfer volume to 15 mL conical tubes and count cells.

21. Plate cells into a 96 well plate (for both ELISA and RT-qPCR). 50,000 cells/well (can also increase to 100,000), 200 total µL volume/well, in triplicate. Use cRPMI+ 50 ng/mL M-CSF.

   i. Treatment groups: i.e. Control, LPS only, LPS+Nigericin (positive control), LPS+Octanal, Octanal only

22. Incubate cells 2 days in TC incubator (37°C, 5% CO₂).

Day 9: Experimental Stimulation

23. Aspirate media, replace with 200 µL fresh cRPMI + 50 ng/ml rhM-CSF.

24. Prepare dilution of LPS stock in cRPMI media (Final concentration in well: 50 ng/mL LPS)

1. Perform a 1:50 dilution of the 0.5 mg/ml stock to get 10 ug/ml LPS.
2. Perform additional 1:10 dilution for a 1 ug/ml working stock. Add 10 ul of the 1 ug/ml stock to designated wells (1:20 dilution to plate).

25. Incubate in TC incubator (37°C, 5% CO₂) for 4 hours.

26. Add Octanal or DMSO (negative control) to wells; 10 µM final concentration for Octanal and 1:1000 DMSO (0.2 µL in 200 µL volume in well).

27. POSITIVE CONTROLS:

1. Nigericin, 5 µM final concentration (stock is 1 mM); needsto be added to LPS primed cells. This is a strong activator of the inflammasome. Nigericin (Product information: Invivogen, Cat # tlrl-nig, 10 mg)

28. Pipet up and down in the wells with P200 multichannel to mix

29. Place in TC incubator (37°C, 5% CO₂) for 8 hrs.

30. After incubation, Spin down plate, 500 x g, 5 min, RT.

31. Transfer all media to 96 well flat bottom polystyrene plate. Seal plate, freeze media in -80°C, until ELISA or CBA analysis.

32. Add 250 µL Trizol.

33. Seal plate and freeze down in -80°C until ready for RNA isolation and RT-qPCR analysis to test the knockdown efficacy.

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