Summary
Macrophage survival assays are a critical component of any Mycobacterium tuberculosis research program. Here we describe the methods that we use routinely for infection of macrophages of various origins. The protocols are efficient, relatively simple and are accepted widely. We provide users with methods for the infection of small numbers of macrophages--more suitable for microscopy; and for larger numbers of macrophages--for flow cytometry analysis or extraction for biochemical characterization.
Keywords: phagocytosis, macrophage, Mycobacterium tuberculosis, infection
1. Introduction
Although we have learned a lot about biology of Mycobacterium spp. that are cultured in broth rich with nutrients, there is a critical need to understand how the pathogen adapts to more natural environments – such as its host macrophage. Macrophages are among the very first cells that will encounter Mycobacterium tuberculosis (Mtb) following its inhalation into the lung and they play crucial role in the outcome of infection. Therefore in vitro models of macrophage infection represent a powerful tool for studying host-pathogen interaction. However it is important to remember that this bacterium is a human pathogen and must be handled under biosafety level 3 containment (see Note 1). Using this model the dynamics of transcriptional response of Mtb to the host environment has been successfully probed (1–3). Through the use of fluorescently labeled reporter Mtb we have been able to look at the localization of bacteria, and the availability of host lipids within the macrophage (4). Flow cytometry and functional physiological assays have enabled us to assess impact of infection on relative phagosomal function and metabolic state (4). Finally we have performed an extensive empirical screen for small molecules capable of impairing the survival of Mtb in its host macrophage and identified many inhibitors that blocked growth in intracellular Mtb but had no effecto on bacteria in rich broth (5). These experimental data all support the contention that macrophage infection and survival assays are critical to expanding our understanding of the biology of the infectious agent as well as its host.
Here we describe the methods that we use for efficient infection of macrophages of differing host origins. One of the biggest technical challenges for these infection assays is the extremely hydrophobic nature of the bacterial cell surface due to its high lipid content. To counteract this we use gelatin-containing infection medium to break up clumps of bacteria without lysing macrophages in addition to the multiple passaging of bacterial suspensions through syringe needles.
We include protocols for both small and large scale infections. The small scale protocols are intended for analysis by methods such as confocal microscopy. Whereas the large scale infection protocols are designed for isolation of macrophages for lipid or protein extraction, bacterial and host RNA and DNA isolation, electron microscopy, flow cytometry or isolation of bacteria after macrophage infection. It is intended that these methods may be modified to accommodate the specific needs of your assay and appropriate scale of choice.
2. Materials
2.1. Cells, Reagents, and Buffers
Macrophages: Bone marrow–derived murine macrophages (BMMØ) are our cells of choice for these assays. BMMØ are derived from the bone marrow extracted from the femur, tibia, and ilium of euthanized mice and maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 1 mM sodium pyruvate, and 10% L-cell conditioned media (BMMØ media). J774 cells (available from the American Type Culture Collection, Rockville, MD) are maintained in DMEM supplemented with 10% FBS and 2 mM L-glutamine + 1 mM sodium pyruvate.
For small-scale protocol: Sterile glass slide with lid and with chambers of preferred format. Typically, 8-well multi-chamber slide with glass coverslip (ibidi, etc.) bottom and with 1 cm2 growth area per well is used if infected macrophages will be examined by confocal microscopy.
For large-scale protocol: Sterile 75 cm2 polystyrene tissue culture flask with filtered cap. Sterile 150 cm2 polystyrene tissue culture flask with filtered cap also can be used (both from Corning etc) (see Note 2).
BMDM medium: DMEM supplemented with 10% fetal calf serum, 10% L-cell conditioned medium, 2 mM L-glutamine, 1 mM sodium pyruvate. Filter-sterilize. Store at 4°C for no longer than 2 months.
HMDM medium: DMEM supplemented with 10% human serum, 2 mM L-glutamine, 1 mM sodium pyruvate. Filter-sterilize. Store at 4°C for no longer than 2 months.
Antibiotics: 100 U/mL penicillin and 100 μg/mL streptomycin.
25 cm2 and/or 75 cm2 polystyrene tissue culture flask with filtered cap or any sterile flask covered with filtered cap that allows to grow up to 10 ml and 40 ml of bacterial culture, respectively, with approximately 50 ml and 230 ml of free space inside the flask.
Middlebrook 7H9 OADC medium: 4.7 g of the Middlebrook 7H9 Broth dehydrated base powder, 2 ml glycerol, 0.05% tyloxapol, 100 mL of Middlebrook OADC Enrichment, 900 mL of distilled water. Filter-sterilize. Store at room temperature.
15 ml conical tube(s).
Basal uptake buffer (BUB): 2.25 g glucose, 2.5 g bovine serum albumin, 0.5 ml gelatin, 50 mg CaCl2, 50 mg MgCl2, 500 ml phosphate-buffered saline (PBS). Filter-sterilize, store at 4°C for no longer than 6 months (see Note 3).
Sterile 1 ml tuberculin syringe with 25 gauge needle.
2.2. Instruments
CO2 incubator.
Spectrophotometer with ability to measure absorbance at 600 nm.
Centrifuge that allows to spin 15 ml-conical tubes at 3,300 × g.
3. Methods
3.1. Macrophage preparation
3.1.1. Small scale, for microscopy
Seed macrophages into glass slide with chambers of the preferred format at about 80% confluency in macrophage media without antibiotics. Typically, 8-well multi-chamber slide with glass coverslip bottom is used to seed 1.8 × 105 murine BMDM in 0.2 ml of macrophage media per well (see Note 4).
Incubate at 37°C and 7% CO2 overnight. Since murine BMDM can still slowly replicate, you need to assume that there is 2 × 105 cells per well on the day of infection.
3.1.2. Larger scale: 75 cm2 or 150 cm2 tissue culture flasks
Here we give an example how to perform an infection of macrophages seeded in one 75 cm2 tissue culture flask. Double all the amounts if you wish to use 150 cm2 tissue culture flask.
Seed 1.5 × 107 macrophages in 20 ml of macrophage media without antibiotics per flask.
Incubate at 37°C and 7% CO2 overnight.
3.2. Preparation of bacterial cultures
Inoculate 8 ml Middlebrook 7H9 OADC medium in a 25 cm2 tissue culture flask with approximately 0.1–0.2 ml of frozen M. tuberculosis glycerol stock.
Incubate at 37°C in standing culture for 7–10 days, until culture reaches logarithmic phase (see Note 5).
Pass into required volume of Middlebrook 7H9 OADC medium depending on scale of infection at starting optical density at 600 nm (OD600) of about 0.05. Use 25 cm2 flasks for volumes not more than 10 ml, and 75 cm2 flasks for volumes up to 40 ml (see Note 6).
Incubate at 37°C in standing culture for 7 days, culture should reach OD600 of approximately 0.6 (see Note 7).
3.3. Infection Protocol
3.3.1. Small scale, for microscopy
Measure OD600 of bacterial culture. Spin down equivalent of 2ml of culture with OD600=0.6 in 15 ml conical tube at 3,300 × g for 10 minutes. For example, if your culture has an OD600 of 0.5, you need to spin down 2.4 ml (0.6*2ml/0.5=2.4ml).
Assuming that OD600=0.6 gives 108 bacteria/ml, you will have 2 × 108 bacteria in the pellet.
Resuspend bacterial pellet in 2 ml of BUB, so that you have 1 × 108 bacteria per ml. Place this suspension in a new 15 ml centrifuge tube (see Note 8).
Using a 1ml tuberculin syringe with 25 gauge needle insert the syringe into the Falcon tube with extreme care and pass the bacterial suspension in and out of the syringe 12–15 times (see Note 9). Proceed to the next step immediately, so that bacteria don’t have chance to clump (see Note 10).
Add required volume of bacterial suspension to the macrophages to achieve desired multiplicity of infection (MOI) (Table 1) (see Note 11). In order to minimize dilution or avoid over-filling the well it is advisable not exceed 40 μl of BUB. If volume is too small to pipette, dilute bacterial suspension as necessary.
Mix bacteria with macrophage media in the well by gentle pipetting up and down 5 times (see Note 12).
Incubate at 37°C and 7% CO2 for 2–5 hours.
Replace media used for infection with 0.22 ml of fresh macrophage media without antibiotics. If assay will exceed two days, change media every other day.
Table 1.
Calculations of bacterial amounts needed to infect macrophages at desired MOI.
| MOI 1:1 | MOI 2:1 | MOI 4:1 | MOI 5:1 | MOI 10:1 | |
|---|---|---|---|---|---|
| Amount of bacteria needed to be added to 2 × 105 cells per well | 2 × 105 | 4 × 105 | 8 × 105 | 1 × 106 | 2 × 106 |
| Volume of bacterial suspension needed to be added to 2 × 105 cells per well, μl | 2 | 4 | 8 | 10 | 20 |
3.3.2. Larger scale: 75 cm2 tissue culture flask
Our calculations are designed to generate an MOI of 4:1. Adjust amounts accordingly if you need to use different MOI.
Measure OD of bacterial culture. Spin down equivalent of 1ml of culture with OD600=0.6 in 15 ml conical tube at 3,300 × g for 10 minutes. For example, if your culture has an OD600 of 0.5, you need to spin down 1.2 ml (0.6*1ml/0.5=1.2ml).
Assuming that OD600=0.6 gives 108 bacteria/ml, you will have 1 × 108 bacteria in the pellet.
Resuspend bacterial pellet in 1.5 ml of BUB. Place this suspension in a new 15 ml Falcon tube in an appropriate holder (see Note 8).
Using a 1ml tuberculin syringe with 25 gauge needle insert the syringe into the Falcon tube with extreme care and pass the bacterial suspension in and out of the syringe 12–15 times (see Note 9). Proceed to the next step immediately, so that bacteria don’t have chance to clump (see Note 10).
Add 3.5 ml of BUB, mix well. Now you have 2 × 107 bacteria per ml.
Transfer 3ml of bacterial suspension into 75 cm2 flask containing macrophages in 20 ml of macrophage media. Mix well by either pipetting up and down multiple times or gentle shaking (see Note 13). Use Table 2 to perform infections at desired MOI.
Incubate at 37°C and 7% CO2 for 2–5 hours.
Replace media used for infection with 20 ml of fresh macrophage media without antibiotics. If assay will exceed two days, change media every other day.
Table 2.
Calculations of bacterial amounts needed to infect macrophages at desired MOI.
| MOI 1:1 | MOI 2:1 | MOI 4:1 | MOI 5:1 | MOI 10:1 | |
|---|---|---|---|---|---|
| Amount of bacteria needed to be added to 1.5 × 107 cells in 75 cm2 flask | 1.5 × 107 | 3 × 107 | 6 × 107 | 7.5 × 107 | 1.5 × 108 |
| Original volume of bacteria at OD600 0.6 needed to be pelleted, ml | 0.5 | 0.5 | 1 | 1 | 2 |
| Total volume of BUB that needs to be added to bacterial pellet, ml | 10 | 5 | 5 | 4 | 4 |
| Concentration of bacteria in BUB suspension, bacteria/ml | 5 × 106 | 1 × 107 | 2 × 107 | 2.5 × 107 | 5 × 107 |
| Volume of bacterial suspension needed to be added to 1.5 × 107 cells in 75 cm2 flask, ml | 3 | 3 | 3 | 3 | 3 |
Acknowledgments
EVN and DGR are supported by the following awards from the National Institutes of Health, USA; AI067027, AI118582 and HL055936 to DGR.
Footnotes
Mycobacterium tuberculosis is a human pathogen and must be handled under Biosafety Level 3 containment by trained personnel. Such facilities have to be maintained under registered institutional safety and employee health monitoring programs. Some investigators use non-pathogenic Mycobacterium spp., such as Mycobacterium smegmatis, as a surrogate but the relevance of data generated with non-pathogenic organisms is open to obvious concerns regarding validity.
In case of 150 cm2 tissue culture flasks double all the amounts described for 75 cm2 tissue culture flask.
If gelatin becomes solidified, pre-warm it at 55–65 °C overnight. Due to gelatin high viscosity, syringe would be the easiest way to measure it out.
If you are using human monocyte-derived macrophages, seed 3 × 105 monocytes per well in media containing antibiotics 7–10 days before infection, change for media without antibiotics on day 7 of incubation.
Cultures can be grown shaking to avoid the generation of hypoxic conditions. In that case, the rate of growth will be twice as fast, so adjust timing accordingly. We prefer to use standing cultures so that the surface glycocalyx of Mtb is less disturbed during culture.
It’s important to pass macrophages after inoculum culture had grown, since the latter quite often contains clumps of bacteria. Therefore avoid transferring these clumps directly into the culture when you perform this procedure.
We recommend using cultures at logarithmic stage for infection, since they are more homogeneous.
Try not to touch sides of the new tube with any part of pipette for safety reasons.
It’s important to use 15 ml conical, since tuberculin syringe fits perfectly inside of it without touching the bottom. When attaching needle to the syringe be careful not to touch the main part of syringe below the plunger to avoid contamination of bacterial suspension. Keep the syringe in the tube throughout this entire process to minimize the potential for self-inoculation. This process can be performed with blunt syringe needles.
If you wish to perform infection with different strains of Mtb, you shouldn’t syringe more than 4 bacterial suspensions before proceeding to the next step to avoid re-clumping of bacteria.
We have noticed that different strains of Mtb behave differently in macrophage infection and induce differing levels of cytotoxicity. When infecting murine BMDM, CDC1551 can be used at higher MOI such as 5 and 10 for 7–10 days of infection without eliminating host cells, whereas H37Rv and Erdman shouldn’t be used at MOI higher than 5, otherwise macrophages will be killed within 3–5 days. In our experience HMDM are less tolerant of Mtb than murine BMDM, so we usually use MOI for CDC1551 not higher than 5 (0.5–3 is preferred), and for Erdman and H37Rv not higher than 1 to maintain infected macrophage monolayers for at least 5 days.
Make sure that you don’t introduce any bubbles in media while pipetting to avoid escape of media due to capillary motion.
If you are infecting multiple flasks, you can mix macrophage media with bacterial BUB-suspension at the same ratios in the separate large flask for all of the samples. Then replace the media in each macrophage-containing flask with this bacterial BUB-macrophage media suspension. Handling the suspension in this manner provides greater consistency between samples.
References
- 1.Schnappinger D, Ehrt S, Voskuil MI, Liu Y, Mangan JA, Monahan IM, Dolganov G, Efron B, Butcher PD, Nathan C, Schoolnik GK. Transcriptional adaptation of Mycobacterium tuberculosis within macrophages: insights into the phagosomal environment. J Exp Med. 2003;198:693–704. doi: 10.1084/jem.20030846. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Rohde KH, Veiga DF, Caldwell S, Balázsi G, Russell DG. Linking the transcriptional profiles and the physiological states of Mycobacterium tuberculosis during an extended intracellular infection. PLoS Pathog. 2012;8:e1002769. doi: 10.1371/journal.ppat.1002769. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Tan S, Sukumar N, Abramovitch RB, Parish T, Russell DG. Mycobacterium tuberculosis responds to chloride and pH as synergistic cues to the immune status of its host cell. PLoS Pathog. 2013;9:e1003282. doi: 10.1371/journal.ppat.1003282. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Podinovskaia M, Lee W, Caldwell S, Russell DG. Infection of macrophages with Mycobacterium tuberculosis induces global modifications to phagosomal function. Cell Microbiol. 2013;15:843–859. doi: 10.1111/cmi.12092. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.VanderVen BC, Fahey RJ, Lee W, Liu Y, Abramovitch RB, Memmott C, Crowe AM, Eltis LD, Perola E, Deininger DD, Wang T, Locher CP, Russell DG. Novel inhibitors of cholesterol degradation in Mycobacterium tuberculosis reveal how the bacterium’s metabolism is constrained by the intracellular environment. PLoS Pathog. 2015;11:e1004679. doi: 10.1371/journal.ppat.1004679. [DOI] [PMC free article] [PubMed] [Google Scholar]