Bacterial Synchronized Transfer Assays in Bone Marrow Derived Macrophages

Merocytophagy (“mero”, Greek for partial; “cytophagy” for cell eating) is a process by which cells acquire microbes and cytosolic material through phagocytosis of a small portion of neighboring cells upon cell-cell contact. Cell-cell contact dependent transfer events can be assessed through co-incubation of differently labeled cells. With these assays, it is difficult to analyze the recipient cells by microscopy or bacterial burden within only recipient cells. Therefore, we established a synchronized transfer assay that allows for recipient cells to be isolated from donor cells following transfer events at a high purity. Here, we present this assay in context of bacterial infections and cytosolic cellular staining. With this protocol, mechanisms of cell-cell contact dependent transfer events and the events following merocytophagy can easily be investigated.

mechanism for the intracellular bacteria to disseminate and sustain infection (Steele et al., 2016 and 2019).
To understand the mechanism of merocytophagy, we established a synchronized transfer assay that can be used to analyze a purified population of recipient cells following transfer events. Previously described transfer assays allow for the quantification of transfer events, whereas this assay allows for the analysis of the engulfed material. Specifically, engulfed material following a transfer event can be analyzed using microscopy or microbial burdens. Herein, we describe a detailed protocol of the synchronized transfer assay to analyze transferred bacteria and cytosolic dye.

1.
Leica Application Suite X (LAS X) (Leica microsystems, www.leicamicrosystems.com), for microscopy analysis Procedure A. L929 cell conditional media preparation

2.
Detach cells by removing existing media and adding 4 ml of TrypLE to flask.

3.
Incubate at 37 °C 5% CO 2 for 5 mins or until cells are completely detached.

4.
Collect detached cells by adding 10 ml of L cells media to flask and transfer to 50 ml conical tube.

5.
Centrifuge at room temperature at 250-300 × g for 5 min to remove TrypLE from media. Re-suspend cells in L cell media.

6.
Count cells by adding 10 μl of trypan blue to 10 μl of cells. Add 10 μl of mixture to hemocytometer and count under light microscope.

7.
Plate cells at a density of 2.5 × 10 5 cells in 75 ml L cell media in T225 flasks.

8.
Rock flasks back and forth to get equal coverage of the entire flask bottom.

10.
Remove media from all of the flasks and place into 50 ml conical tubes.

12.
To ensure thorough mixing of all collected media, combine all of the supernatants into one 50 ml conical tube.

13.
Vacuum filter media through a 250 ml filter unit.

15.
Transfer tubes to −20 °C for long-term storage. Thaw media at 37 °C when needed for bone marrow macrophage differentiation.

B. Bone marrow derived macrophages preparation
Note: Protocol can be performed using J774A.1 macrophage (ATCC® TIB-67), if bone marrow derived macrophages are unavailable. If performing EEA-1 staining, do not allow cells to fix for more than 7 min or antibody will not stain well.

1.
Euthanize mouse by CO 2 and cervical dislocation in accordance with IACUC protocol.

2.
Remove femurs from mouse in a biosafety cabinet.
Note: Keep bones intact and clean bones of any excess tissue.

3.
For each femur, cut each end using sterile scissors. Cut approximately 1 mm off of each head of the bone or until you see an opening large enough for a 26G needle.

4.
With forceps, hold the femur over a 50 ml conical tube.

5.
Prepare a 10 ml syringe with needle. Fill syringe with 10 ml of DMEM.

6.
Insert needle into the opening of the bone.

7.
Flush femur with 5 ml of DMEM into the 50 ml conical tube.
Note: Be sure to stick the needle into each opening of the femur. If needle cannot enter the femur, cut more off of the end.

9.
Filter the solution with a 40 μm cell strainer into a fresh 50 ml conical tube to remove clumps of tissue.

11.
Remove media and gently resuspend pellet in residual media (approximately 1 ml).

15.
Remove media and resuspend cells in approximately 10 ml of L cell differentiation media (Recipe 1 and Procedure A).

16.
Seed approximately 5 ml in 20 ml final volume of media into non-tissue culture treated Petri dish.
Note: Ensure proper differentiation by checking morphology periodically. See Figure 1 for reference of proper differentiation (cells appear with elongated shape).

18.
On Day 6, remove media from cells and wash cells by pipetting 10 ml of 1x DPBS (with Mg and Ca). Repeat wash 2 more times.

20.
Incubate plates at 37 °C for approximately 5 min or until cells begin to lift off.

23.
Remove media and resuspend cells in 10 ml of DMEM with 10% FBS.
Note: Expect a cell count of 6 to 8 million cells per plate.

5.
Next day, spin down 2 ml of culture at room temperature at 20,000 × g for 2 min.

6.
Remove the supernatant and resuspend each pellet in 1 ml of PBS.

7.
Blank OD spec with 1 ml of PBS.

8.
Measure OD 600 of culture (desired OD should be 0.34-0.36, if not incubation should continue).

11.
Add 10 μg/ml of gentamicin in a total volume of 500 μl per well.

12.
Twenty hours following inoculation of bacteria, replace media on all samples.

13.
Invert coverslip of recipient cells onto donor cells to allow for direct contact.

15.
Remove the coverslip and put it into the original well in its original orientation.

16.
At desired time point, add 4% PFA to coverslip (1 ml per well) and incubate at room temperature for 7 min (for EEA-1 staining) or 10 min for other antibodies.

20.
Mount coverslips onto slides using mounting media by adding approximately 10 uμl of mounting media to slides and inverting coverslip onto slide.

Data analysis
Microscopy results were collected using a Leica DM400 upright fluorescence microscope. For each sample, 50 images were taken at each desired time point per experiment. Images were analyzed using Leica LAS X software. The number of cells that acquired material from donor cells can be determined by counting the number of recipient cells that obtained either bacteria or CFSE (Figure 2