Materials and Reagents

1. Serological Pipette (e.g., Thermo Scientific, catalog number: 14-387-163)
2. 250 ml filter unit, 0.2 micron PES membrane filter, sterile (VWR, catalog number: 10040-464)
3. 10 ml syringes (Fisher Scientific, catalog number: 201635)
4. Fisherbrand^TM Easy Reader^TM Conical Polypropylene centrifuge tube 15 ml (Fisher Scientific, catalog number: 05-539-12)
5. Fisherbrand^TM Easy Reader^TM Conical Polypropylene centrifuge tube 50 ml (Fisher Scientific, catalog number: 05-539-13)
6. Fisherbrand^TM Microscope Slides, Frosted (Fisher Scientific, catalog number: 12-552-3)
7. Fisherbrand^TM Non-Tissue Culture treated Petri Dishes with Clear Lid (Fisher Scientific, catalog number: FB0875712)
8. Serological Pipette Tips (Various Sizes) (Fisher Scientific, catalog numbers: 07-200-571, 07-200-574, 07-200-573, 07-200-575)
9. Fisherbrand^TM Cell Strainer (40 μM) (Fisher Scientific, catalog number: 22363547)
10. Matsunami Micro Cover Glasses, Round (18 mm diameter) (VWR^TM, catalog number: 10026-138)
11. Micropipette Tips (Various Sizes) (VWR, catalog numbers: 10017-062, 10017-064, 10017-66, 1007-068, 1007-092)
12. Corning Costar Flat Bottom Cell Culture Plates (Fisher Scientific, catalog number: 3513)
13. 26G BD Precision Glide needles (Fisher Scientific, catalog number: 148266A)
14. C57BL/6J Mice (Jackson Laboratory, Stock No. 000664)
15. Fetal Bovine Serum (FBS) (Atlanta Biologicals, catalog number: S10350)
16. Francisella tularensis subsp. holarctica inducible T6SS mutant (Steele et al., 2019)
17. Francisella tularensis subsp. holarctica live vaccine strain (LVS) (Center for Disease Control, CDC)
18. Recombinant murine M-CSF (rmM-CSF) (Peprotech, catalog number: 315-02)
19. Remel^TM Hemoglobin (Fisher Scientific, catalog number: R451402)
20. J774A-1 Macrophages (ATCC^® TIB-67)
21. L929 fibroblasts (ATCC^® CLL-1)
22. Gibco^TM TrypLE Express Enzyme (1x) (Fisher Scientific, catalog number: 12-605-028)
23. L-Aspartic Acid (Arcos Organics, catalog number: 56-84-8)
24. DL-Isoleucine (Fisher Scientific, catalog number: A17521)
25. L-Leucine (Arcos Organics, catalog number: 12512-100)
26. DL-Methionine (Arcos Organics, catalog number: 59-51-8)
27. DL-Serine (Fisher Scientific, catalog number: A15184)
28. L-Tyrosine (Fisher Scientific, catalog number: J63511)
29. L-Cysteine (Sigma-Aldrich, catalog number C7602-25G)
30. Thiamine HCl (Arcos Organics, catalog number: 67-03-8)
31. Spermine HCl (Fisher Scientific, catalog number: 5677-5GM)
32. L-Arginine (Arcos Organics, catalog number: 74-79-3)
33. L-Histidine (Arcos Organics, catalog number: 71-00-1)
34. L-Lysine (Fisher Scientific, catalog number: 657-27-2)
35. L-Proline (Fisher Scientific, catalog number: A10199)
36. DL-Threonine (Fisher Scientific, catalog number: A10606)
37. DL-Valine (Arcos Organics, catalog number: 516-06-3)
38. Ammonium Chloride (NH₄Cl) (ACROS Organics, catalog number: AC423285000)
39. 4% Paraformaldehyde (PFA) (Fisher Scientific, catalog number: AAJ19943K2)
40. Bacto Agar (Fisher Scientific, catalog number: DF1040010)
41. Brain Heart Infusions (BHI, Fisher Scientific, catalog number: DF0037178)
42. Cell Trace Far Red (CTR) Dye (ThermoFisher, catalog number: C34564)
43. Cell Trace CFSE (CFSE) (ThermoFisher, catalog number: C34553)
44. Corning^TM RPMI 1640 Medium (Mod.) 1x with L-Glutamine (Fisher Scientific, catalog number: MT10-040-CV)
45. Dulbecco’s Modification of Eagles Medium (DMEM) with 4.5 g/L glucose, L-glutamine, sodium pyruvate (Corning Life Sciences, catalog number: 10-013-CM)
46. DPBS (with magnesium (Mg) and calcium (Ca) (Corning Life Sciences, catalog number: 21-030-CM)
47. DPBS (without magnesium (Mg) and calcium (Ca) (Corning Life Sciences, catalog number: 21-031-CM)
48. EDTA disodium salt (ACROS Organics, catalog number: AC409975000)
49. Gibco^TM GlutaMAX^TM Supplement (Fisher Scientific, catalog number: 35050-061)
50. Gibco^TM Sodium Bicarbonate 7.5% solution (Fisher Scientific, catalog number: 25080-094)
51. Gibco Penicillin-Streptomycin (10,000 U/ml) (Fisher Scientific, catalog number: 15140-148)
52. HyClone^TM MEM Non-essential Amino acids (Fisher Scientific, catalog number: SH30238.01)
53. HyClone^TM Sodium pyruvate (Fisher Scientific, catalog number: SH30239.01) 
54. Potassium Bicarbonate (KHCO₃) (Fisher Scientific, catalog number: P184-500)
55. Sodium Hydroxide (Fisher Scientific, catalog number: SS255-1)
56. Trypan Blue (MP Biomedicals, catalog number: 195532)
57. Vectashield Antifade Mounting Medium with DAPI (Vector Laboratories, catalog number H-1200)
58. Calcium Pantothenate (Arcos Organics, catalog number: 137-08-6)
59. Magnesium Sulfate (MgSO₄·7H₂O) (Arcos Organics, catalog number: 10034-99-8)
60. Iron (II) Sulfate Heptahydrate (FeSO₄·7H₂O) (Fisher Scientific, catalog number: A15178)
61. Potassium Phosphate Monobasic (KH₂PO₄) (Fisher Scientific, catalog number: P285-500)
62. Potassium Phosphate Dibasic Anhydrous (K₂HPO₄) (Fisher Scientific, catalog number: P288-100)
63. D-Glucose (Fisher Scientific, catalog number: A16828)
64. HCl
65. NaCl
66. Isovitalex
67. 10% FBS DMEM (see Recipes)
68. ACK lysis buffer (see Recipes)
69. Chocolate agar plates (see Recipes)
70. Chamberlain’s defined medium (CDM) (Chamberlain et al. [1965]) (see Recipes)
71. L cell media (see Recipes)
72. Bone marrow differentiation media (see Recipes)
73. 0.5 M EDTA (see Recipes)
74. 10 mM EDTA in 1x DPBS (without Mg and Ca) (see Recipes)
75. 10x Glucose Salts (see Recipes)
    

Equipment

1. Blunt forceps (Fisher Scientific, catalog number: 08-875-8B)
2. T75 flasks (Fisher Scientific, catalog number: 130190)
3. T175 flasks (Fisher Scientific, catalog number: 130191)
4. Surgical Scissors (Fisher Scientific, catalog number: 08-951-5)
5. Micropipettes (e.g., Rainin, catalog numbers: 17008655, 17008650, 17008652, 17008653)
6. Benchtop Swing Bucket Centrifuge (e.g., Eppendorf, catalog number: 022625004)
7. Hemocytometer (e.g., Fisher Scientific, catalog number 02-671-6）
8. Shaking Water bath (e.g., Fisher Scientific, catalog number: S37368)
9. Humidified 5% CO₂ incubators set to 37 °C (e.g., NuAire, NU-5700) 
10. Leica DM4000 upright fluorescence microscope
11. OD spectrometer 
12. Light Microscope
13. Biosafety cabinet
    

Software

1. Leica Application Suite X (LAS X) (Leica microsystems, www.leica-microsystems.com), for microscopy analysis

Procedure

1. L929 cell conditional media preparation
   1. Grow L929 fibroblasts to confluency in two T75 flasks using L cell media (Recipe 9). Incubate at 37 °C 5% CO₂ for approximately 2 days.
   2. Detach cells by removing existing media and adding 4 ml of TrypLE to flask.
   3. Incubate at 37 °C 5% CO₂ 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 x 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 x 10⁵ 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.
   9. Incubate at 37 °C 5% CO₂ for 10-12 days (watch for cobblestone appearance of cells).
   10. Remove media from all of the flasks and place into 50 ml conical tubes.
   11. Centrifuge at 300 x g for 5 min at 4 °C.
   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.
   14. Aliquot 45 ml into 50 ml conical tubes.
   15. Transfer tubes to -20 °C for long-term storage. Thaw media at 37 °C when needed for bone marrow macrophage differentiation.
       
       
       
2. 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 CO₂ 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.
   8. Repeat Steps B6 and B7 with second femur.
   9. Filter the solution with a 40 μm cell strainer into a fresh 50 ml conical tube to remove clumps of tissue.
   10. Centrifuge cells at room temperature at 250-300 x g for 5 min.
   11. Remove media and gently resuspend pellet in residual media (approximately 1 ml).
   12. Add 1 ml of ACK lysis buffer (Recipe 2) and incubate for 60 s.
   13. Add 9 ml of DMEM.
   14. Centrifuge cells at room temperature at 250-300 x g for 5 min.
   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.
   17. Allow cells to differentiate in 37 °C 5% CO₂ incubator for 6 days.
       Note: Ensure proper differentiation by checking morphology periodically. See Figure 1 for reference of proper differentiation (cells appear with elongated shape).
       
       
       Figure 1. Morphology of Day 5 differentiated BMDMs. Appearance of elongated shape as indicated by arrowheads.
       
       
   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.
   19. Following final wash, add 3-4 ml/plate of 10 mM EDTA in 1x DPBS (without Mg and Ca), pH 7.2-7.4.
   20. Incubate plates at 37 °C for approximately 5 min or until cells begin to lift off.
   21. Add 5-8 ml/plate of DMEM with 10% FBS DMEM (Recipe 5) and transfer to a 15 ml conical tube.
   22. Centrifuge cells at room temperature at 250-300 x g for 5 min.
   23. Remove media and resuspend cells in 10 ml of DMEM with 10% FBS.
   24. Count cells using a hemocytometer.
       Note: Expect a cell count of 6 to 8 million cells per plate.
   25. Seed a 12-well tissue culture treated plate at 2 x 10⁵ cells per well in a total volume of 1 ml per well.
       Note: For each sample, seed 1 well with a coverslip (recipient sample) and 1 well without a coverslip (donor cells).
   26. Incubate overnight at 37 °C 5% CO₂.
       
       
       
3. Transfer of cell permeable dye
   Note: Wells should be set up with CFSE and CTR cells only (i.e. cells that do not have contact).
   1. Stain donor cells with Cell Trace CFSE (CFSE) at a final concentration of 5 μM in dPBS with Mg and Ca (500 μl per well) in a 12-well plate.
   2. Stain recipient cells with Cell Trace Far Red (CTR) at a final concentration of 1 μM in dPBS with Mg and Ca (500 μl per well) in a 12-well plate.
   3. Incubate cells at 37 °C 5% CO₂ for 20 min.
   4. Wash cells 3 times each with 1 ml of pre-warmed 10% FBS DMEM.
      Note: 10% FBS DMEM should be pre-warmed in a 37 °C water bath for 20 min.
   5. After the final wash, pick up the coverslip with recipient cells and invert it onto the well with donor cells so that the cells are in direct contact.
   6. Incubate for 30 min at 37 °C 5% CO₂.
   7. Remove the coverslip and return it into the original well in its original orientation.
   8. 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.
   9. Wash coverslips 3 times with 1 ml of 1x DPBS (with Mg and Ca). 
   10. Perform desired microscopy staining (e.g., LAMP-1 [Steele et al., 2019]).
   11. Wash coverslips 3 times with 1 ml of 1x DPBS.
   12. Mount coverslips onto slides using mounting media by adding approximately 10 μl of mounting media to slides and inverting coverslip onto slide.
       
       
       
4. Bacterial infection
   Note: Various bacteria can be used for this assay with modifications needed for growth and infection conditions. Control wells should be set up with CFSE and CTR cells only.
   1. Streak out Francisella tularensis freezer stock on chocolate agar.
   2. Incubate plate at 37 °C for 3 days.
   3. Pick 4-6 individual colonies from the plate and suspend colonies in 3 ml of CDM.
   4. Incubate culture overnight at 37 °C in a shaking water bath.
   5. Next day, spin down 2 ml of culture at room temperature at 20,000 x 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 OD₆₀₀ of culture (desired OD should be 0.34-0.36, if not incubation should continue).
   9. Infect donor cells at MOI 100 in 250 μl total volume of 10% FBS.
   10. Incubate for 2 h at 37 °C 5% CO₂. 
   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.
   14. Incubate for 30 min at 37 °C.
   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.
   17. Wash coverslips 3 times with 1 ml of 1x DPBS (with Mg and Ca). 
   18. Perform desired microscopy staining (e.g., LAMP-1 [Steele et al., 2019])
   19. Wash coverslips 3 times with 1 ml of 1x DPBS.
   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 for reference). Transwells (or physical separation) can be used to determine the number of phagocytosis events of extracellular material (Steele et al., 2016 and 2019).


Figure 2. Representative microscopy of CFSE transfer events. A. CFSE stained donor BMDMs. B. CTR stained recipient BMDM. C. CFSE transfer to CTR stained recipient BMDM.

Recipes

1. Bone marrow differentiation media (150 ml for 1 mouse)
   75 ml of DMEM
   30 ml of heat inactivated FBS
   45 ml L929 conditional media (can be substituted with M-CSF, Griffin et al., 2013)
   1.5 ml of 100 mM L-glutamine (or GlutaMax)
   1.5 ml of 100 mM sodium pyruvate
   1.5 ml of 7.5% sodium bicarbonate solution
   Mix and filter sterilize
2. ACK lysis buffer
   8.3 g of NH₄Cl
   1.0 g of KHCO₃
   200 μl of 0.5 M EDTA
   Add ddH₂O to 1 L, adjust to pH 7.2-7.4 and filter sterilize
3. 0.5 M EDTA
   Dissolve 186.1 g EDTA disodium salt in 750 ml of ddH₂O
   Add 15 g solid NaOH to solution to solubilize EDTA
   Use 5 N NaOH to adjust pH to 7.5-8.0
4. 10 mM EDTA in 1x DPBS (without Mg and Ca)
   Add 20 ml of 0.5 M EDTA to 1 L of 1x DPBS (without Mg and Ca)
5. 10% FBS DMEM
   Add 100 ml of thawed heat inactivated FBS to 1 L of DMEM
6. Chamberlain’s defined medium (CDM) (Chamberlain et al., 1965)
   Add components to a final concentration of 1x in a desired volume of H₂O:
   L-Aspartic Acid (100x stock: 2 g into 50 ml HCl)
   DL-Isoleucine (100x stock: 2 g into 50 ml HCl)
   L-Leucine (100x stock: 2 g into 50 ml HCl)
   DL-Methionine (100x stock: 2 g into 50 ml HCl)
   DL-Serine (100x stock: 2 g into 50 ml HCl)
   L-Tyrosine (100x stock: 2 g into 50 ml HCl)
   L-Cysteine (100x stock: 1 g into 50 ml HCl)
   Thiamine HCl (100x stock: 200 mg into 50 ml HCl)
   Spermine HCl (100x stock: 200 mg into 50 ml HCl)
   L-Arginine (100x stock: 2 g into 50 ml H₂O)
   L-Histidine (100x stock: 1 g into 50 ml H₂O)
   L-Lysine (100x stock: 2 g into 50 ml H₂O)
   L-Proline (100x stock: 5 g into 50 ml H₂O)
   DL-Threonine (100x stock: 2.5 g into 50 ml H₂O)
   DL-Valine (100x stock: 2 g into 50 ml H₂O)
   Calcium Pantothenate (100x stock: 100 mg into 50 ml H₂O)
   10x Glucose Salts (see Recipe 7)
   Once all components are added, pH solution to 6.2-6.4
7. 10x Glucose salts
   25 g NaCl
   338 mg MgSO₄·7H₂O
   5 mg FeSO₄·7H₂O
   2.5 g KH₂PO₄
   2.5 g K₂HPO₄
   10 g Glucose
   250 ml H₂O
8. Chocolate agar plates
   In a 2 L flask, add:
   25 g of BHI
   15 g of Bacto Agar
   500 ml of H₂O
   In a second 2 L flask add:
   10 g of hemoglobin
   500 ml of H₂O
   Autoclave for 20 min on liquid cycle
   Remove media from autoclave and place into a 50 °C water bath
   Once cooled, add 10 ml of isovitalex
   Combine hemoglobin to BHI and gently stir by hand
9. L cell media
   500 ml of RPMI 1640
   50 ml of heat inactivated FBS
   2.5 ml of Pen/strep
   5 ml of 100 mM sodium pyruvate
   5 ml 100 mM non-essential amino acids
   

Acknowledgments

The authors would like to thank the National Institute of Allergy and Infectious Diseases (NIH) for funding (R01AI082870 and R56AI139476).

Competing interests

No competing interests declared.

Ethics

All animals were handled according to approved institutional animal care and use committee (IACUC) protocol #4946 at Washington State University.

References

1. Ahmed, K. A., Munegowda, M. A., Xie, Y. and Xiang, J. (2008). Intercellular trogocytosis plays an important role in modulation of immune responses. Cell Mol Immunol 5(4): 261-269.
2. Chamberlain, R. E. (1965). Evaluation of live tularemia vaccine prepared in a chemically defined medium. Appl Microbiol 13: 232-235.
3. Griffin, A. J., Crane, D. D., Wehrly, T. D., Scott, D. P. and Bosio, C. M. (2013). Alternative activation of macrophages and induction of arginase are not components of pathogenesis mediated by Francisella species. PLoS One 8(12): e82096.
4. Ireton, K. (2013). Molecular mechanisms of cell-cell spread of intracellular bacterial pathogens. Open Biol 3(7): 130079.
5. Steele, S. P., Chamberlain, Z., Park, J. and Kawula, T. H. (2019). Francisella tularensis enters a double membraned compartment following cell-cell transfer. Elife 8: e45252.
6. Steele, S., Radlinski, L., Taft-Benz, S., Brunton, J. and Kawula, T. H. (2016). Trogocytosis-associated cell to cell spread of intracellular bacterial pathogens. Elife 5: e10625.