Culturing splenic, hepatic and alveolar macrophages

A leukotriene-dependent spleen-liver axis drives TNF production in systemic inflammation

Monique T. Fonseca, Eduardo H. Moretti, Lucas M. M. Marques, Bianca F. Machado, Camila

F. Brito, Jady T. Guedes, Evilin N. Komegae, Thayna S. Vieira, William T. Festuccia, Norberto

P. Lopes, Alexandre A. Steiner*

*Corresponding author. Email: asteiner@usp.br

Published 20 April 2021, Sci. Signal.14, eabb0969 (2021) DOI: 10.1126/scisignal.abb0969

Detailed protocol for splenic macrophage culture

1. Transcardial perfusion and organ harvesting
   1. Anesthetize the rat with isoflurane (5% for induction; 2.0 to 2.5% for maintenance).
   2. Open the thoracic cavity and perform a transcardial perfusion (right atrium cut) with 50 ml of ice-cold PBS (phosphate-buffered saline) at a rate of 10 ml/min.
   3. Dissect out the entire spleen and keep it in ice-cold HBSS (Hanks' Balanced  Salt Solution).
2. Organ dissociation
   1. In a sterile environment, place the organ on a Petri dish containing 2 mL of ice- cold HBSS, and repeatedly pinch it with serrated forceps until it is dissociated into fine pieces.
   2. Transfer the dissociated contents along with the HBSS to a conical tube, and complete the volume to 12 ml with ice-cold PBS.
   3. Centrifuge for 10 min at 600 x g and 4°C.
   4. Discard the supernatant.
3. Gross purification of macrophages
   1. Resuspend the pellet in 3 ml of ACK lysing buffer (Gibco, cat. No. A10492-01), and incubate for 3 min at room temperature.
   2. Centrifuge for 10 min at 600 x g and 4°C.
   3. Discard the supernatant.
   4. Resuspend the pellet in 12 ml of ice-cold PBS.
   5. Centrifuge for 10 min at 600 x g and 4°C.
   6. Discard the supernatant.
   7. Resuspend the pellet in 3 ml of ice-coldHBSS.
   8. In a 15 ml conical tube, make a three-phase gradient of Percoll (Sigma-Aldrich, cat. No. P1644), each phase of 3 ml. Phase 1 consists of 30% Percoll in HBSS (tinted with phenol red), phase 2 of 40% (not tinted), and phase 3 of 50% (tinted with phenol red). Tinting helps with visualization of interfaces.
   9. Via the tube wall, slowly add the cell suspension obtained in 3g on top of the Percoll gradient.
   10. Centrifuge for 20 min at 400 x g and 4°C, with the centrifuge breaks off.
   11. The cloud enriched in macrophages should be formed between the layers containing 30% and 40% of Percoll.
   12. Using a pipette, transfer the cloud to a 15-ml conical tube, and complete the volume to 12 ml with ice-cold PBS.
   13. Centrifuge for 10 min at 400 x g and 4°C.
   14. Discard the supernatant.
   15. Wash and re-centrifuge (400 x g, 4°C, 10 min) the pellet twice with 10 ml of ice- cold HBSS.
   16. Discard the supernatant.
   17. Resuspend the pellet in 1 ml of ice-cold AIM V medium (Gibco, cat. no. 12055091).
4. Cell counting and plating
   1. Estimate the number viable cells per milliliter of the suspension obtained in 3q. We use Trypan Blue exclusion as an indicator of cell viability and a Neubauer chamber for counting.
   2. Add more AIM V medium (this time warm, 37ºC) to the cell suspension in order to reach a cell density of 1 x 10⁷ live cells/ml.
   3. Transfer 1 ml of the cell suspension to each well of a 6-well, flat-bottom polystyrene culture plate.
   4. Add an additional 1 ml of medium to eachwell.
   5. Incubate overnight under standard culture conditions (37ºC and 5% CO₂).
   6. Early on the next day, gently aspirate as much of the medium as possible.
   7. Wash the wells with 1 ml of warm (37°C) PBS, then aspirateit. Steps 4f and 4g are intended to remove non-adherent cells.
   8. Refill each well with 2 ml of warm (37°C) AIM V medium.
   9. Perform the experiment at the desired culture condition.
   10. In our hands, these cultures are viable for experiments at 37ºC and 5% CO₂ for 6-8 h after the overnight incubation.

Detailed protocol for Kupffer cell culture

1. Liver perfusion and harvesting
   1. Anesthetize the rat with isoflurane (5% for induction; 2.0-2.5% for maintenance).
   2. Access the abdominal cavity via a midline laparotomy and isolate the portal vein.
   3. Via the portal vein (with the vena cava cut downstream of the liver), perfuse the liver with 40 ml of warm (37ºC) PBS (phosphate-buffered saline) at a rate of 5 ml/min. This is intended to clear the organ from circulating leukocytes without detaching Kupffer cells from the inner walls of the sinusoids.
   4. Harvest all lobes of the liver and immediately place them in ice-cold HBSS (Hanks' Balanced Salt Solution).
2. Organ dissociation
   1. In a sterile environment, use a serrated forceps to break down the harvested organ into small pieces at room temperature. This can be done on a Petri dish.
   2. Split the resulting pieces into 10 vials, each filled with 1 ml of HBSS containing type IV collagenase at 1 mg/ml (Sigma-Aldrich, cat. no. C5138), DNase at 0.05%  (Worthington, cat. no. LS002006) and fetal bovine serum at 5%.
   3. Incubate at 37ºC for 15 min under constant stirring. We used a dry incubator (Thermomixer) for that, with stirring at 400 rpm.
   4. With the help of the barrel of a 3-ml syringe, pass the contents of each tube through a cell strainer with pore of 100 µm.
   5. Transfer the filtrate of all vials to a single 50-ml conical tube.
   6. Complete the volume to 40 ml with ice-coldHBSS containing 5% of fetal bovine serum. This should be done promptly to inactivate the digesting enzymes.
   7. Centrifuge for 10 min at 400 x g and 4°C.
   8. Discard the supernatant.
   9. Resuspend the pellet in 12 ml of ice-cold HBSS containing 5% of fetal bovine serum.
   10. Centrifuge for 10 min at 400 x g and 4°C.
   11. Discard the supernatant.
   12. Resuspend the pellet in 12 ml of ice-cold HBSS containing 5% of fetal bovine serum.
   13. Centrifuge for 10 min at 400 x g and 4°C.
   14. Discard the supernatant.
   15. Resuspend the pellet in 3 ml of ice-coldHBSS containing 5% of fetal bovine serum.
3. Gross purification of macrophages
   1. In a 15 ml conical tube, use a transfer pipette to gently add 3 ml of the cell suspension obtained in step 2o over 3 ml of Ficoll-Paque PLUS (GE Healthcare, cat. no. GE17- 1440-02). The addition should be done slowly through the tube wall to avoid mixing of the two phases.
   2. Centrifuge for 20 min at 400 g and 4°C, with the centrifuge breaks off.
   3. A cloud (enriched in macrophages) should be formed at the interface of the two phases.
   4. Using a pipette, transferthe cloud to a 15 ml conicaltube and completethe volume to 12 ml with ice-cold PBS.
   5. Centrifuge for 10 min at 400 x g and 4°C.
   6. Discard the supernatant.
   7. Resuspend the pellet in 1 ml of ice-coldAIM V medium (Gibco, cat. no. 12055091).
4. Cell counting and plating
   1. Estimate the numberviable cells per milliliter of the suspension obtained in 3g. We use Trypan Blue exclusion as an indicator of cell viability and a Neubauer chamber for counting.
   2. Add more AIM V medium (this time warm, 37ºC) to the cell suspension in order to reach a cell density of 2 x 10⁶ live cells/ml.
   3. Transfer 1 ml of the cell suspension to each well of a 12-well, flat-bottom polystyrene culture plate.
   4. Incubate for 2 h under standardculture conditions (37ºC and 5% CO₂).
   5. Gently aspirate as much of the medium as possible.
   6. Wash the wells with 1 ml of warm (37°C) PBS, then aspirate it. Steps 4e and 4f are intended to remove non-adherent cells.
   7. Refill each well with 1 ml of warm (37°C) AIMV medium.
   8. Perform the experiment at the desiredculture condition.
   9. In our hands, thesecultures are viablefor experiments for 6-8 h under standardculture conditions (37ºC and 5% CO₂).

Detailed protocol for alveolar macrophage culture

1. Bronchoalveolar lavage
   1. Anesthetize the rat with isoflurane (5% for induction; 2.0 to 2.5% formaintenance).
   2. Open the thoracic cavity and excise the lungs along with the trachea.
   3. Perform a bronchoalveolar lavage via the trachea, flushing the lungs in and out 10 times with ice-coldPBS (phosphate-buffered saline).Perform each flush with 10 ml of PBS, gently massaging the lungs.
   4. The flushes typically yield ~70 ml of lavage fluid, which should be divided into two 50-ml conical tubes.
2. Cell Gross purification of macrophages
   1. Centrifuge the lavage fluid for 10 min at 600 x g and 4°C.
   2. Discard the supernatant.
   3. Resuspend the pellet in 2 ml of ACK lysing buffer (Gibco, cat. no. A10492-01) and incubate for 3 min at room temperature.
   4. Centrifuge for 10 min at 600 x g and  4°C
   5. Discard the supernatant.
   6. Resuspend the pellet in 1 ml of ice-cold AIM V medium(Gibco, cat. no. 12055091).
3. Cell counting and plating
   1. Estimate the number viable cells per milliliter of the suspension obtained in 2f. We use Trypan Blue exclusion as an indicator of cell viability and a Neubauer chamber for counting.
   2. Add more AIM V medium (this time warm, 37ºC) to the cell suspension in order to reach a cell density of 1 x 10⁶ live cells/ml.
   3. Transfer 1 ml of the cell suspension to each well of a 12-well, flat-bottom polystyrene culture plate.
   4. Incubate for 2 h under standard cultureconditions (37ºC and 5% CO₂).
   5. Gently aspirate as much of the medium as possible.
   6. Wash the wells with 1 ml of warm (37°C) PBS, then aspirateit. Steps 3e and 3f are intended to remove non-adherent cells.
   7. Refill each well with 1 ml of warm (37°C) AIM V medium.
   8. Perform the experiment at the desired culture condition.
   9. In our hands, these cultures are viable for experiments for 26-28 h.

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