Lentiviral Vector Preparation for Efficient Gene and MicroRNA Modulation of Peritoneal Cavity Tissue-Resident Macrophages In Vivo in Mice

All animal work was conducted in accordance with Institutional and UK Home Office guidelines.

NOTE: All in vivo studies with lentivirus should be performed according to local and national guidelines on the ethical use of animals in research, as well as adhering to all regulations associated with the use of category II infectious materials. Animal welfare should also be monitored in accordance with local regulations. In this step of the protocol, extreme care needs to be taken when working with lentiviral particles and sharps.

1. Preparation of HEK293T cells for transfection

NOTE: Perform these steps under a sterile tissue culture biological safety cabinet.

1. Prepare complete Dulbecco's Modified Eagle Medium (cDMEM) for HEK293T cells by combining the 450 mL of DMEM with 10% (v/v) fetal calf serum (50 mL) and a final concentration of 100 U/mL penicillin/streptomycin.
2. Defrost HEK293T cells at least 1 week prior to planned transfection. Maintain healthy growing conditions and passage every 2-3 days using trypsin + EDTA to detach the cells from the flask.
3. One day prior to transfection, carefully aspirate the growth medium from the cells and gently wash cells with sterile DPBS. Add 1-5 mL of trypsin to the flask and incubate at 37 °C in a 5% CO₂ incubator for 2-5 min.
4. Add 5-10 mL of cDMEM and spin the cells at 350 x g for 5 min. Remove the liquid and resuspend the cell pellet in 10 mL of cDMEM. Count the cells and seed 10-11 x 10⁶ viable HEK293T cells per T175 flask in 20 mL of cDMEM.
5. Incubate at 37 °C in a 5% CO₂ incubator overnight to allow the HEK293T cells to reach 70%-80% confluency.
6. The following day, confirm the confluency of cells under a light microscope.
7. Remove the media from the flask with a 25 mL serological stripette without disturbing the cell monolayer.
8. Gently add 10 mL of DPBS and rock the plate to wash the cells, taking care not to disturb the cell monolayer.
9. Remove DPBS with a 25 mL serological stripette.
10. Add 15 mL of new cDMEM on the wall per T175 flask so as not to disturb the cell monolayer, and return the plate to the incubator at 37 °C.

2. Transfection of HEK293T cells using non-liposomal lipid transfection reagent

1. In a 15 mL centrifuge tube, prepare the lentiviral components: 2 µg lentiviral plasmid, 1.5 µg pΔ8.91 (pCMV-Δ8.91), and 1 µg pMD2.G (pCMV-MD2.G) with Buffer EC (Transfection reagent kit) to a final volume of 600 µL.
2. Add 36 µL of enhancer solution. Mix the components using a 1 mL pipette by repeatedly sucking up a part of the liquid and putting it back drop-by-drop directly on the remaining solution. Leave for 5 min at room temperature (RT).
3. Add 120 µL of transfection reagent and mix as above approximately 20 times. Incubate for 10 min at RT.
4. Add 5.2 mL of cDMEM and mix as above with a 5 mL serological stripette.
5. Using a disposable transfer pipette, add the transfection mix dropwise directly onto the HEK293T cells monolayer (avoiding the flask walls), spreading the mix at different spots in the flask.
6. Distribute the plasmid by gently rocking the flask from side to side. Avoid getting any liquid in the flask lid.
7. Incubate the flask at 37 °C in a 5 % CO₂ incubator for 48 h. Confirm effective transfection of HEK293T cells by the appearance of a fluorescent marker, here GFP, within 24 h of transfection monitored under a cell imaging fluorescent microscope.
   NOTE: For constructs without the fluorescent marker, HEK293T cells must be tested for the presence of the used marker gene or by expression change in the gene/protein of interest by appropriate techniques, e.g., qPCR. Alternatively, successful transfection can be indirectly verified during testing of the lentivirus collection on Jurkat cells at the later stages of the protocol by the above techniques.
   ​CAUTION: Transfected HEK293T cells are considered infectious, and appropriate safety precautions should be implemented when handling these cells. Local rules should be followed, which could typically include working under a category II biology safety cabinet, the use of double gloves, and appropriate decontamination solution for disinfection, for example, increased concentration of waste bleach (2,000 ppm) or equivalent solution according to institutional biosafety guidelines. All solutions and plastics coming in contact with lentivirus preparation should be disinfected according to institutional biosafety procedures.

3. Collection of lentiviral particles

1. Prepare decontamination solution, a high concentration (2,000 ppm) bleach (sodium hypochlorite) solution, or equivalent agent according to institutional biosafety guidelines in a bucket and place it in the biology safety cabinet.
2. Prepare the biology safety cabinet by removing any excess items, such as empty tube racks etc.
3. Pre-label a 50 mL centrifuge tube per T175 HEK293T cells flask and remove the lid from the centrifuge tube. Place the tubes in a stable rack.
4. Retrieve the flask from the incubator and confirm GFP expression using a fluorescent microscope with a 488 nm laser (Figure 1A). The intensity of the signal depends on the transfection efficiency of the procedure and used constructs.
5. Collect the media from the cells into the pre-labeled 50 mL centrifuge tube. Tilt the T175 HEK293T flask with transfected cells so the medium collects in the bottom corner of the flask. Using a 25 mL serological stripette, collect the medium without disturbing the cell monolayer. Some floating cells might be visible at this point. Transfer the medium to a clean 50 mL centrifuge tube and close the lid.
   1. Using a 25 mL serological stripette, add 25 mL of fresh, warm cDMEM to the flask. Make sure to direct the medium flow on the walls of the flask not to disturb the cell monolayer. Return the flask with transfected HEK293T cells to the incubator (37 °C, 5% CO₂).
      NOTE: A second collection of lentivirus and further purification can be performed after additional 24 h following the procedure outlined in the steps above.
6. When lentivirus collection is completed after 24 h or 48 h, add decontamination solution to the cells, ensuring it covers the cell monolayer. Keep the flask horizontal for the next 24 h, then discard it following appropriate category II regulations.

4. Purification of lentivirus

1. Filter collect medium from step 3.5.
   1. Remove the plunger from the 50 mL syringe and insert a low protein binding polyether sulfone or polyvinylidene fluoride 0.45 µm sterile filter into the syringe end. Using a 25 mL serological stripette, add the collected medium from step 3.5.1 to the syringe and gently return the plunger.
   2. Push the plug slowly to pass the medium through the filter into a fresh 50 mL centrifuge tube. If the flow reduces significantly, position the syringe with the filter pointing upwards and pull out the plunger enough to clear the medium from the filter.
   3. Carefully, replace the filter on the syringe with a new one and dispose of the used filter in the decontamination solution. Continue medium filtration. When finished, decontaminate the filter and the syringe by submerging the filter and filling the syringe with the decontamination solution.
2. Pre-cool the ultracentrifuge by setting the temperature to 4 °C and shut the lid to allow the temperature to acclimate.
3. Add 3 mL of 20 % sucrose solution (20% w/v in ultrapure water, filter sterilized using a 0.22 µm filter) into the bottom of the conical ultracentrifuge tube.
4. Using a 25 mL serological stripette, overlay filtered medium on top of the sucrose layer, being careful not to disturb the layers.
   1. Use the lowest speed setting on the pipette boy. Angle the conical ultracentrifuge tube to about 45° and slowly add the filtered lentivirus-containing medium from step 4.1 to the tube wall. Ensure that the medium and sucrose do not mix, and clear separation of the layers is visible (Figure 1B). As the volume of the medium layer increases, slowly tilt the ultracentrifuge tube back to an upright position.
5. If the total volume, including the sucrose and the medium, in the ultracentrifuge tube is less than 29 mL, top up with fresh cDMEM to avoid the tube collapsing during the spin. For multiple T175 mL collections, mix filtered medium preparations and use multiple ultracentrifuge tubes. Top up the final ultracentrifuge tube with the medium as required.
6. Ensure the ultracentrifuge buckets are clean; there is no medium residue on the bottom of the bucket or the caps. If required, wipe out with ~70 % alcohol. Put appropriate tube adapters in the bottom of each bucket (Figure 1C) and gently lower the ultracentrifuge tubes into the buckets.
7. Close the buckets securely and hang them on the allocated spaces on the spin-out rotor.
8. Ensure that the buckets are balanced with the same volumes of sucrose and medium. A spin-out rotor must never be run without buckets, although opposing buckets may be left empty²⁰ (Figure 1D).
9. Carefully insert the rotor into the ultracentrifuge and turn on the vacuum. Set the ultracentrifuge acceleration and deceleration rate to the lowest setting, and run the lentivirus preps at 85,000 x g for 90 min at 4 °C.
10. Wait for the ultracentrifuge to reach the required speed (about 3-5 min) before walking away to ensure the rotor is inserted properly and the spin will not terminate.
11. While the ultracentrifuge is running, clean up the workspace according to category II safety requirements and prepare the space for the next steps.
12. When the spin finishes, disable the vacuum and carefully remove the rotor so as not to disturb the samples.
13. Investigate the ultracentrifuge for any spills and confirm no leaks from the buckets. In case of spillage, double glove and decontaminate the centrifuge and external surface of buckets with antiviral products.
14. Remove the buckets from the ultracentrifuge and carefully transport the buckets to the category II biology safety cabinet.
    NOTE: Lentivirus particles collect at the bottom of the ultracentrifuge tube. The pellet is not visible to the naked eye.
15. Pour the sucrose and medium with one smooth motion directly into the waste container with the decontamination solution.
16. Keeping the ultracentrifuge tube inverted, transfer it to the double layer of tissue and leave it to dry for 10 min. Meantime, wipe the inside of the buckets with water-soaked tissue and air-dry upside down.
17. Carefully dry any remaining liquid from the rim of the ultracentrifuge tube with tissue before turning it upright. Disinfect the tissue and the surface underneath.
18. Resuspend the virus pellet in 1 mL of serum-free media or solution required for further experimentation by gently pipetting the solution up and down. Leave it for 15 min at RT inside the category II biology safety cabinet.
19. Gently mix the lentivirus preparations using 1 mL pipette before aliquoting into 1.5 mL screw top tubes (e.g., cryotubes). Prepare the aliquots for the in vivo work (multiplicity of 100 µL) and for lentivirus titer in Jurkat T cells (1 x 20 µL) (see step 5).
20. Avoid freeze-thaw cycles; lentiviral preparations can be stored at -80 °C for up to 6 months without a negative effect on infectivity.

5. Titration of lentivirus production in Jurkat T cells

1. Prepare complete Roswell Park Memorial Institute 1640 medium (cRPMI-1640) for Jurkat T cells by supplementing 500 mL of RPMI-1640 with 10% (v/v) fetal calf serum and final concentration of 100 U/mL penicillin/streptomycin.
2. To ensure a healthy culture of Jurkat T cells, maintain the cells in culture for up to 1 week before infection.
3. On the day of the lentivirus titration, plate out viable Jurkat T cells in a 24-well plate at 2 x 10⁵ cells/well in 200 µL per well of cRPMI-1640.
4. Use freshly collected lentivirus or thaw the lentivirus vial containing 20 µL on ice and mix gently by pipetting up and down.
5. Using serial dilution in cRPMI-1640, infect Jurkat T cells with 0.25 µL, 0.5 µL, 1 µL, 2.5 µL, 5 µL, and 10 µL of lentivirus stock. Gently rock the plate to ensure equal distribution of the lentivirus. Non-infected Jurkat T cells serve as a negative control.
6. Incubate the plate at 37 °C. After 4 h, top up each well with cRPMI to a total volume of 400 µL. Return the plate to the incubator at 37 °C for 3 days.
7. After 48 h post-infection, confirm GFP expression under a fluorescent imaging system.
8. 3 days after infection, collect each well of the 24-well plate into separate 1.5 mL collection tubes and centrifuge the cells at 350 x g at 4 °C for 5 min.
9. Discard the supernatant. Resuspend the pellet in 300 µL of 2% paraformaldehyde (PFA) prepared to 2% (w/v) in DPBS and leave it for 15 min on ice in the dark.
10. Centrifuge the cells at 350 x g at 4 °C for 5 min and resuspend the pellet in fluorescence-activated cell sorting (FACS) buffer (sterile DPBS + 4% FCS + 1 mM sterile EDTA).
11. Analyze the GFP expression frequency and mean fluorescent intensity of the lentivirus infected and its control cells using flow cytometry (Figure 2).

6. In vivo lentivirus infection of tissue-resident peritoneal macrophages

1. In a category II biological safety cabinet, load insulin needles with a total volume 200 µL of serum-free media medium containing the required amount of lentivirus (use single-use needles, 30 G for animal welfare and to avoid fluid loss in the needle).
2. Place the needle sheath back on the needle using the one-hand scoop technique. Keep the needle on ice and inject within 30 min.
3. In the animal facility, set up a category II biological safety cabinet prior to injections (Figure 1E).
   1. Lay down a sheet of clean tissue. Loosen the sheath on the insulin needle containing lentivirus.
   2. Prepare a Petri dish containing small pieces of tissue and chlorhexidine gluconate-based disinfectant, a 50 mL tube containing decontamination solution (2,000 ppm bleach solution), and a sharp safe container.
4. Restrain the mouse by grasping the skin at the nape of the neck²¹. Properly restrained mouse is immobile, and this is required for safety.
5. With the abdomen facing up, point the animal's head slightly down. Inject the lentivirus Intraperitoneally into the lower right quadrant of the abdominal cavity. This is to avoid injection into any peritoneal cavity organs.
6. Before releasing the mouse, fill the syringe with decontamination solution and safely dispose of it in the sharp safe box.
7. Wipe the injection site on the abdomen of the mouse with tissue soaked in disinfectant and return the animal to the cage.
8. House the lentivirus-injected mouse in category II scantainer or individually ventilated cages (IVC) (isolated cages with high-efficiency air filtration) for a minimum of 72 h after injection. Place an appropriate information card on the front of the cage.
9. Move the mouse to the new category I holding cage after 72 h post-infection, depending on local safety approvals. Monitor mice daily for a total of 3 days from injection.

7. Collection of peritoneal cells from lentivirus-infected mouse

CAUTION: For collections within 72 h post lentivirus injection, follow institutional category II biological safety rules. Bedding and holding cage where infected animals were kept in the first 72 h post lentivirus injection must be decontaminated according to institutional category II biological safety rules.

1. Euthanize mice following institutional regulations, for example, by inhalation of increasing concentration of CO₂, followed by confirmation of death by cervical dislocation.
2. Clean the abdomen of the mouse with 70% isopropanol and carefully cut the skin to expose the peritoneal cavity membrane.
3. Lavage the peritoneal cavity with 6 mL of ice-cold FACS buffer using a 10 mL syringe and 23 G needle. Avoid puncturing any organs.
4. Gently massage the peritoneum to dislodge cells to the FACS buffer.
5. Collect the peritoneal fluid using the same syringe and needle.
6. Remove the needle and transfer the cells to a 15 mL centrifuge tube.
7. Keep the peritoneal lavages on ice.
8. Collect other required organs and store them as appropriate to the study.
9. Dispose of animal carcasses according to local animal and safety guidelines.

8. Peritoneal cell staining and analysis

1. Centrifuge the peritoneal lavage at 350 x g at 4 °C for 5 min, discard the supernatant in the decontamination solution, and resuspend the cells in 1 mL of FACS buffer.
2. Count the collected cells and plate 4 x 10⁵ cells per well in a V-bottom 96-well plate.
3. Perform viability staining using a fixable reagent (e.g., Fixable Near-IR Dead Cell Stain Kit used here) according to manufacturer instructions.
4. If cells were infected within the last 72 h, fix the cells in 2% PFA for 15 min on ice. Add an equal volume of cold DPBS and centrifuge again at 350 x g at 4 °C for 5 min.
5. Prepare the blocking buffer: Mix 4 µg/mL 2.4G2 antibody in 10% (v/v) rat serum in FACS buffer for surface staining or permeabilization buffer for intracellular staining.
6. Resuspend each well containing cell pellet in 50 µL of blocking buffer and incubate at 4 °C for 15 min.
7. Prepare antibody mix in either 50 µL of FACS buffer (for surface staining) or permeabilization buffer (for intracellular staining) per each sample. The final staining volume for each sample will be 100 µL, including 50 µL of blocking buffer. Calculate the antibody concentrations accordingly (Table 1).
8. Add 50 µL of antibody mix to samples and 50 µL of isotype controls and control buffer to control samples. Incubate the samples for 30 min on ice in the dark. Include unstained cells and isotype controls as required.
9. Wash each well with 100-200 µL of ice-cold DPBS and centrifuge the plate at 350 x g at 4 °C for 5 min. Repeat this step to wash away any unbound antibodies. Analyze the samples on a flow cytometer.

9. Extraction of cells from organs

1. Prepare 1 mL of digestion mix per organ: Mix Hank's balanced salt solution (HBSS), 2 mg/mL collagenase type IV, and 0.03 mg/mL DNase I (include 1.5 mg/mL of hyaluronidase for lung digestion).
2. Transfer the collected organ to 1 mL of digestion mix and mince it with scissors.
3. Filter the cells through a 40 µm strainer and centrifuge at 350 x g at 4 °C for 5 min.
4. If isolating cells from the lung, spleen, or liver, lyse red blood cells using ACK lysis buffer (150 mM NH₄Cl, 10 mM KHCO₃, 0.1 mM Na₂EDTA pH = 7.4). Filter the cells through a 40 µm strainer and centrifuge at 350 x g at 4 °C for 5 min.
5. Stain and analyze the cells as described in section 8.