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Macrophages are key cells involved in orchestrating host defense against infections. Here, we describe the protocol for a bacterial killing assay in macrophages that can be adapted to any bacterial pathogen. Using this assay, we analyzed the survival of wild-type and mutant strains of Escherichia coli (E. coli) within RAW 264.7 cells, a widely used macrophage cell line. Bacterial mutants defective in intracellular survival within macrophages can be delineated using this assay.

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Gentamicin Protection Assay to Determine Bacterial Survival within Macrophages

Microbiology > Antimicrobial assay > Killing assay
Authors: Sargurunathan Subashchandrabose
Sargurunathan SubashchandraboseAffiliation: Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, USA
For correspondence: sarguru@umich.edu
Bio-protocol author page: a1651
 and Harry L.T. Mobley
Harry L.T. MobleyAffiliation: Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, USA
Bio-protocol author page: a1652
Vol 4, Iss 18, 9/20/2014, 5311 views, 0 Q&A, How to cite
DOI: http://dx.doi.org/10.21769/BioProtoc.1235

[Abstract] Macrophages are key cells involved in orchestrating host defense against infections. Here, we describe the protocol for a bacterial killing assay in macrophages that can be adapted to any bacterial pathogen. Using this assay, we analyzed the survival of wild-type and mutant strains of Escherichia coli (E. coli) within RAW 264.7 cells, a widely used macrophage cell line. Bacterial mutants defective in intracellular survival within macrophages can be delineated using this assay.

Materials and Reagents

  1. RAW 264.7 cells (ATCC, TIB-71)
  2. E. coli CFT073 or other strains of bacteria
  3. LB broth (Beckton Dickinson, catalog number: 244610)
  4. Agar (Beckton Dickinson, catalog number: 214010)
  5. RPMI1640 (Life Technologies, catalog number: 11875-093)
  6. Gentamicin 50 mg/ml (Life Technologies, catalog number: 15750-102)
  7. PBS (pH 7.4)
  8. LB plates (see Recipes)
  9. Certified, heat inactivated fetal bovine serum (FBS) (Life Technologies, catalog number: 10082139) (see Recipes)
  10. 100x penicillin, streptomycin and glutamine (PSG) (Life Technologies, catalog number: 10378-016) (see Recipes)
  11. Saponin (Sigma-Aldrich, catalog number: 8047-15-2) (see Recipes)

Equipment

  1. 24 well plates (Corning, Costar®, catalog number: 3524)
  2. 0.22 micron filter (Millipore, catalog number: SCGP00525)
  3. 37 °C, 5% CO2 incubator
  4. Sorvall Legend RT table top centrifuge
  5. Shimadzu UV-1601PC Spectrophotometer

Procedure

  1. Culture RAW 264.7 cells in RPMI1640 with 10% heat inactivated fetal bovine serum and 1x penicillin streptomycin and glutamine at 37 °C in a CO2 incubator.
  2. Count the cells using standard procedures and seed 2 x 105 RAW cells/well, by adding 1 ml per well in a 24 well plate. Set up 2 plates; one for T0 and another for T2 and incubate in a CO2 incubator.
  3. Start overnight cultures of CFT073 in 3 ml of LB broth in a 10 ml polypropylene tube and incubate at 37 °C shaking at 200 rpm. Use appropriate culture media when using other bacterial strains.
  4. Determine the multiplicity of infection (MOI = number of bacteria per RAW 264.7 cell) and alter the inocula preparation accordingly. For an MOI of 10, add 2 x 106 cfu of E. coli CFT073 for a well containing 2 x 105 RAW cells.
  5. Pellet the bacterial cells from the 3 ml culture by centrifuging at 4,000 rpm for 10 min in a Sorvall centrifuge. Resupend the pellet in 3 ml PBS.
  6. Adjust the OD600 of broth culture to 4 in PBS, by measuring the optical density of a 1:10 diluted culture in a spectrophotometer and calculating the optical density of undiluted culture.
  7. Dilute 1:100 in PBS.
  8. Dilute 1:10 in RPMI 1640 without antibiotics.
  9. Wash RAW cells twice with PBS.
  10. Add 1 ml of diluted culture to each well; three technical replicates per sample and three biological replicates of the entire experiment are recommended. Use the same set up for T0 and T2 plates.
  11. Incubate at 37 °C, 5% CO2 for 30 min.
  12. Aspirate supernatant and wash 3 times with PBS.
  13. Add 1 ml of RPMI 1640 with gentamicin (200 µg/ml) per well.
  14. Incubate the T0 plate at 37 °C, 5% CO2 for 15 min.
  15. Incubate the T2 plate at 37 °C, 5% CO2 for 2 h.
  16. After 15 min or 2 h, remove the supernatants entirely by aspiration.
  17. Lyse cells with 1 ml of filter sterilized 1% saponin in H2O per well by incubating for 10 min at room temperature followed by vigorous pipetting. Determine the extent of lysis using a standard inverted microscope.
  18. Plate undiluted and -2 dilution for T0 and T2 time points on LB plates.
  19. % Killing = [(T0-T2)/T0]*100

Recipes

  1. LB plates
    Add 15 g of agar to 1 L of LB broth
  2. FBS and PSG
    Added at 10% and 1x final concentration, respectively, to RPMI1640
  3. Saponin
    Dissolved in autoclaved double distilled water and filter sterilized using a 0.22 μM filter

Acknowledgments

This protocol was adapted from the publication: Subashchandrabose et al. (2013).

References

  1. Subashchandrabose, S., Smith, S. N., Spurbeck, R. R., Kole, M. M. and Mobley, H. L. (2013). Genome-wide detection of fitness genes in uropathogenic Escherichia coli during systemic infection. PLoS Pathog 9(12): e1003788.


How to cite this protocol: Subashchandrabose, S. and Mobley, H. L. (2014). Gentamicin Protection Assay to Determine Bacterial Survival within Macrophages. Bio-protocol 4(18): e1235. DOI: 10.21769/BioProtoc.1235; Full Text



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