Materials and Reagents

RAW 264.7 murine macrophage cell line (MΦs)
E. coli strains, marked with constitutive expression of yellow (YFP) and cyan (CFP) fluorescent proteins (e.g. E. coli- MC4100, galK::CFP/YFP, Amp^RStrep^R)
RPMI media1640 (Life Technologies, Gibco^®, catalog number: 11875-093 )
2-mercaptoethanol solution (Life Technologies, Invitrogen^TM, catalog number: 31350-010 )
1 M HEPES buffer (Life Technologies, Invitrogen^TM, catalog number: 15630-056 )
100 mM sodium pyruvate (Life Technologies, Invitrogen^TM, catalog number: 11360-039 )
Heat-inactivated fetal bovine serum (FBS)
200 mM L-glutamine (Life Technologies, Gibco^®, catalog number: 25030-081 )
10,000 U/ml penicillin/streptomycin (Life Technologies, Gibco^®, catalog number: 15140-122 )
Streptomycin sulfate salt (Sigma-Aldrich, catalog number: S9137 )
50 mg/ml Gentamycin solution (Sigma-Aldrich, catalog number: G1397 )
1x Phosphate-buffered saline (PBS)
Trypan blue solution (Sigma-Aldrich, catalog number: T8154 )
CpG-ODN -1826 (Sigma-Aldrich) (5´-TCCATGACGTTCCTGACGTT-3´)
RPMI-complete media (see Recipes)
RPMI-strep media (see Recipes)

Equipment

37 °C, 5% CO₂ cell culture incubator
Microscope
Centrifuge (Sigma-Aldrich, model: 4K15 with a rotor 11150)
Neubauer cell counting chamber
Air flow chamber
12-well microtiter plates
Cell culture flasks (25 cm² and 75 cm² growth area)
15 ml Falcons tubes
Cell scrapers
2 μm size beads (Sphero AccuCount Blank Particles)
Flow cytometer

Procedure

Grow MΦs (in the 37 °C, 5% CO₂ incubator) in 75 cm² culture flask in RPMI- complete media until 80% confluency (Note 1).
Change old media (remove old media by aspiration and add fresh RPMI-complete media) and detach Mϕs with a cell scraper.
Carefully pipette culture up and down until pellet is dissolved.
Centrifuge in 15 ml Falcon tubes at 1,200 rpm for 5 min (Note 2).
Remove supernatant and re-suspend pellet in the same volume of RPMI-Strep media (Note 3).
Count MΦs in the Neubauer cell counter with Trypan blue dye (use 10 μl of cell suspension with 10 μl of dye). Allow the dye to stain for approximately 5 to 15 min. If cells are exposed to extended period of time to this dye, viable cells, as well as nonviable cells, may begin to uptake dye.
Approximately 0.7-1.3 x 10⁶ cells/ml activate with CpG-ODN at a final concentration of 2 µg/µl for 24 h in 10 cm² flask (8 ml total volume) (Note 4).
Grow separately evolved E. coli - CFP (or YFP) and ancestral E. coli - YFP (or CFP) bacterial populations from the frozen stock in RPMI-Strep media (3 ml of the total volume for each well) in the 12-well plates for 24 h (for the evolved E. coli - CFP (or YFP) please see our other protocol “Evolution of Escherichia coli to Macrophage Cell Line” (Miskinyte and Gordo, 2014).
After 24 h repeat steps 2-6, but for the step 4 use 1,000 rpm instead of 1,200.
Seed 0.8-1.6 x 10⁶ cells/ml in 12-well microtiter plates (3 ml total volume for each well).
Allow cells to attach in the 37 °C, 5% CO₂ incubator for 2 h.
Mix E. coli - CFP (evolved) with E. coli - YFP (ancestral) at a ratio of 1 to 1.
Count numbers of CFP (Ni_e) and YFP (Ni_a) bacteria before competition in the mixture by using 2 μm size beads in the flow cytometer (Note 5).
Wash MΦs with RPMI-Strep media (remove old media by aspiration and add fresh RPMI-Strep media).
Infect MΦs at the multiplicity of infection (MOI) of 1 to 1 (mixture of 10⁶ bacteria/ml to 10⁶ MΦs/ml).
After 24 h of infection, detach MΦs with a cell scraper and centrifuge culture at 4,000 rpm for 10 min. This procedure lyses MΦs and releases intracellular bacteria (Note 6).
Discard the supernatant and resuspend the pellet in 3 ml of PBS.
Dilute bacteria in PBS and count CFP (Nf_e) and YFP (Nf_a) bacterial numbers after competition in Flow cytometer.
Use Formula 1 to calculate relative fitness increase of the evolved bacteria over the ancestral.

Formula 1. Selection coeficient, a measure of relative fitness increase. S_coef is the selective advantage of the evolved strain e over the ancestral strain a, Nf_e and Nf_a are the numbers of evolved (e) and ancestral (a) bacteria after competition and Ni_a and Ni_e are the initial numbers, before the competition.

Notes

You should compare the amount of space covered by cells with unoccupied spaces to estimate approximately percent confluency (check: http://www.abcam.com/ps/pdf/protocols/cell culture.pdf).
If the MΦ culture has many dead (aggregated or detached cells) centrifuge at 800 rpm. This way healthy cells will pellet at the bottom of the tube and dead cells will remain mainly in the supernatant.
Streptomycin is used to reduce the possibility of contamination. The strain of E. coli is STR resistant. If you use a different antibiotic, test for its toxicity on the MΦs cell line.
This dilution is necessary because MΦs will grow in 24 h. We use CpG-ODN-1826, because ODN 1826 is a B-class CpG ODN specific for mouse TLR9 that leads to strong immunostimulatory effects after 24 h of activation. If you intend to use other cell line (e.g. ODN 2395 is specific for class C human TLR9) choose an appropriate ODN.
You can use the following dilution to count in the flow cytometer: 10 µl of bacterial culture (diluted 1: 100), 10 µl of beads and 180 ml PBS.
You can also detach MΦs, by bending a blue 1,000 µl pipette tip and using it as a scraper. Remember that for each replicate well you need to use a new sterile scraper.

Recipes

RPMI-complete media
500 ml RPMI 1640
50 ml FBS
5 ml sodium pyruvate
5 ml HEPES
5 ml L-glutamine
0.5 ml 2-mercaptoethanol solution
0.5 ml gentamycin solution
5 ml penicillin/streptomycin solution
Stored at 4 °C
RPMI-strep media
500 ml RPMI 1640
50 ml FBS
5 ml sodium pyruvate
5 ml HEPES
5 ml L-glutamine
0.5 ml 2-mercaptoethanol solution
1 ml of 50 mg/ml streptomycin solution
Stored at 4 °C

Acknowledgments

This protocol was adapted or modified from Richard E. Lenski, Michael R. Rose, Suzanne C. Simpson and Scott C. Tadler 1991, Long-Term Experimental Evolution in Escherichia coli. I. Adaptation and Divergence During 2,000 Generations. The American Naturalist, Vol. 138, No. 6, pp. 1315-1341. The research received funding from the European Research Council under the European Community's Seventh Framework Programme (FP7/2007–2013)/ERC grant agreement no 260421 – ECOADAPT. IG acknowledges the salary support of LAO/ITQB & FCT.

References

Lenski, R. E., Rose, M. R., Simpson, S. C. and Tadler, S. C. (1991). Long-term experimental evolution in Escherichia coil. I. Adaptation and divergence during 2,000 generations. American Naturalist 138:1315-1341.
Miskinyte, M. and Gordo, I. (2014). Evolution of Escherichia coli to macrophage cell line. Bio-protocol 4(17): e1227.
Miskinyte, M., Sousa, A., Ramiro, R. S., de Sousa, J. A., Kotlinowski, J., Caramalho, I., Magalhaes, S., Soares, M. P. and Gordo, I. (2013). The genetic basis of Escherichia coli pathoadaptation to macrophages. PLoS Pathog 9(12): e1003802.