Using Enhanced Green Fluorescence Protein-expressing Escherichia Coli to Assess Mouse Peritoneal Macrophage Phagocytosis

This protocol demonstrated a simple and producible method to assess phagocytosis ability of macrophages using EGFP-expressing Escherichia coli. Hello, my name is Jun-Yu from The Second Hospital of Dalian Medical University. Today we are going to show you an easy and visualize the way to assess macrophage phagocytosis which can be easily done within two hours.

This method was widely used in the study of the innate immune function. It is believed that innate immune function remained intact in the elderly. So today we are going to isolate the peritoneal macrophages from the aged and young mice and to see the phagocytic ability of these two groups.

Well, let's get started. Firstly, synthesize the EGFP gene fragment and clone the fragment using high-fidelity Taq DNA polymerase. Then ligate the PCR product into the pET SUMO vector.

Thirdly, transform the ligation products into the E.coli strain and induce EGFP expressing with lactose. These EGFP-expressing E.coli served as the marker for the phagocytosis assay. Next, a mouse peritoneum macrophages were isolated and cultured.

Then, EGFP-expressing E.coli were coincubated with the macrophages for one hour at 37 degrees centigrees. After the quenching step, the macrophages were ready for assessment by both fluorescence microscope and flow cytometer. Synthesize the EGFP gene fragment and amplify the fragment with a forward and reverse primers using high-fidelity Taq DNA polymerase.

To ensure that the PCR products had single three-end adenine overhangs for TA cloning in the next step, a 30-minute extension at 72 degrees centigrees after the last cycle is recommended. Check the PCR product by agarose gel electrophoresis. If the fragment was amplified correctly, a 717 bp band can be observed on the gel.

Clone the PCR product into the pET SUMO vector using the TA cloning method with T4 DNA ligase. Incubate the reaction at room temperature for 30 minutes. Add five microliter of PCR product into 100 microliter of BL21 competent cells.

Heat shock the cells at 42 degrees centigrees for 90 seconds, then keep the mixture on ice for three minutes. Add 400 microliter of LB medium, preheated at 37 degrees centigrees. Shaking one hour at 37 degrees centigrees and 120 rpm.

Inoculate the bacteria onto the surface of LB plate with 100 microgram per milliliter kanamycin to screen the vector-transformed E.coli. Incubate the plate in the 37 degrees centigrees oven overnight. On the next day, inoculate a positive colony into five milliliter of LB media with 100 microgram per milliliter kanamycin.

Incubate in the 37 degree centigrees shaking incubator at 120 rpm for two hours. Then add the inducer lactose to a final concentration of 0.5 millimole per liter and continue to shaking six hours, inducing EGFP expression. Empirically, when shaking six hours, the OD600 may reach 0.7 or higher.

Finally, using fluorescence microscope to verify the extent of EGFP expressing. Add 10 microliter of the bacterial culture medium to a slide. Then cover with a cover slip.

Examine the expression of EGFP under the fluorescence microscope. Add 3.5 gram of thioglycollate into 100 milliliter of distilled water. Sterilize in autoclave before use.

Aspirate the thioglycollate medium into the one-milliliter sterile syringe in the hood. One mouse per syringe to avoid the infection. Inject one milliliter of 3.5%thioglycollate medium into the mouse peritoneal cavity using a 23-gauge needle and maintain the mouse with water and food ad libitum for three days.

After three days, euthanize the mouse by cervical dislocation after rapidly inducing anesthesia by sevoflurane in a closed box. Then, put the mouse into a dish with 75%ethanol to sterile and transfer into the hood quickly. Place the mouse on the plate and pin the front paw on the board to fix the mouse position.

Using a five-milliliter syringe with a 20-gauge needle, inject five milliliter of cold PBS at the lower abdomen into the mouse peritoneal cavity, avoiding puncturing the bowel. Perform a gentle massage on two sides of the mouse abdomen. Then aspirate the abdominal fluid gently and slowly.

Dispense the peritoneal fluid into a 50-milliliter centrifuge tube. Repeat these steps two or three times. Centrifuge the suspended cells for 10 minutes at 400 g in four to eight centigrade.

Discard the supernatant and resuspend the cell pellet in RPMI 1640 medium with 10%fetal bovine serum. Add five million cells into each well of the six-well plate for the flow cytometry assay and 500, 000 cells per well into a 24-well plate for fluorescence microscope. Culture the cells at 37 degrees centigrees in a 5%carbon dioxide incubator overnight.

The culture medium can be refreshed after three hours to remove nonadherent cells because most of these were lymphocytes. Observe the cells under a bright field microscope to evaluate the cell viability and cell density. The image shown here was in normal condition of the primary cell.

Usually, the macrophage cell density was not high, because major part of the peritoneal cells was lymphocytes. Remove the culture medium from the 24-well plate. Add 100 microliter of fresh culture medium and 10 microliter of bacterial medium.

Then incubate the plate at 37 degrees centigrees in a 5%carbon dioxide incubator for one hour. Wash gently with 500 microliter of cold PBS per well three to five times to wash out non-internalized bacteria. Incubate the cells with 4%formaldehyde in PBS at room temperature for 30 minutes.

Wash the cells with PBS three times. Add phalloidin 633 conjugate working solution to stain F-actin. Store in a dark, humid place at room temperature for 60 minutes.

Add DAPI working solution to stain the cell nuclear and incubate for five minutes in a dark place at room temperature. Rinse once with PBS and once with the same volume in distilled water. The EGFP-expressing E.coli displayed in green served as the marker of phagocytosis.

To minimize experimental errors and make proper interpretation of the results, set groups and control tubes for the experiment as listed in Table 2. For the control group which will be placed on the ice, remove the medium from the six-well plate and wash with PBS once. Then add 70 millimole per liter cold EDTA one milliliter into the well to detach the cells and transfer into the flow cytometry tube.

Add 50 microliter of bacterial suspension into the tube and place it on the ice for one hour. For the other groups, remove the culture medium, add one milliliter fresh medium into each well. Add 50 microliter of bacteria suspension into the wells according to the group setting as described in table two.

Then place the six-well plate in the 37 degree centigrees 5%carbon dioxide incubator for one hour. To quench the fluorescence of non-internalized E.coli, add 200 microliter of 0.8%crystal violet water solution into the well and sway shortly. This step intended to avoid false positive result by the EGFP E.coli binding to the surface of the macrophages but not internalized.

Wash the cells with PBS three times to remove any residual crystal violet. Then add 70 millimole per liter cold EDTA one milliliter into the well to detach the cells and transfer into the flow cytometry tube. Centrifuge the tubes 2, 000 rpm five minutes and discard the supernatant.

Add 100 microliter of PBS to resuspend the cells. Add five microliter of F4 ATP conjugated antibody into the tubes or isotype according to the group setting. Vortex briefly and incubate the samples on ice for five to 10 minutes in the dark.

Add one milliliter PBS into each tube and centrifuge 2, 000 rpm five minutes. Discard the supernatant. Resuspend the cell pellets with 200 to 300 microliter of PBS for flow cytometry analysis.

Run each tube and acquire data for at least 10, 000 events of F4/80-positive cells. Here are the fluorescence images of peritoneal macrophages from the young and aged groups. The red fluorescence represents F-actin.

The green represents EGFP-expressing E.coli, and the blue represents nucleus stained by DAPI. These images suggest that macrophages from the young mouse had a stronger phagocytic ability than those from the aged mouse. The F4/80-positive and EGFP-positive cells indicated the phagocytic ability of the macrophages.

These results are consistent with the trend of fluorescence microscope results. Well, you see, although the number of the innate immune cells may preserved in aged mouse, the phagocytic ability decreased significantly compared to the young mouse. Many methods have been employed to assess macrophage phagocytosis.

Here we demonstrate an improved method which is convenient, quick, and economically feasible. With EGFP-expressing E.coli, a phagocytosis assay can be easily performed and measured by both flow cytometer and fluorescent microscope according to the researcher's prefers. Also this method directly measures the phagocytic ability.

The results are more reproducible than other indirect methods.