Preparation of Digestion-Tracking Bacteria
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New Protocol
Materials:
- One of:
- RFP BL21 E. coli (RC-105, kanamycin resistant)
- GFP BL21 E. coli (RC-108, ampicillin resistant)
- GFP + RFP BL21 E. coli (RC-110, kanamycin + ampicillin resistant)
- RFP+smURF BL21 E. coli (RC-094, kanamycin resistant)
- LB medium
- 1,000X stocks of the appropriate antibiotic(s)
- 50 mg/mL kanamycin
- 100 mg/mL ampicillin
- 1,000X stock of IPTG (1 M, 0.238g/mL)
- 25 mM stock solution of biliverdin if using RC-094 or another smURF containing vector
Preparing the Bacteria:
- Prepare ~2 mL of LB + the appropriate antibiotic(s) at a 1X concentration in a 14 mL snap-cap tube.
- Inoculate with a loop of the desired bacteria from the frozen stock.
- Grow overnight, 37°C, shaking.
- Prepare 2 mL of fresh LB + sufficient antibiotics for 2.5 mL of medium in a 14 mL snap-cap tube.
- Inoculate this new tube with 400 µL of the overnight culture.
- Grow for 1 hr, 37°C, shaking.
- Add 2.5 µL IPTG to the culture.
- If using a smURF-containing bacteria, add 2.5 µL (25 µM) biliverdin at this time.
- Incubate overnight, 37°C, shaking.
- Make a 1:10 dilution of the bacteria into LB with a minimum volume of 150 µL. Use this to measure the OD600 of the culture (see Table 1).
- Remove 1 x 108 bacteria from this culture, dilute to 1 mL in PBS, and pellet by centrifugation. Remove the supernatant, resuspend in 1 mL of PBS, and repellet.
- Suspend the bacterial pellet in 700 µL of PBS, and add to this 300 µL of 16% PFA. Incubate at room temperature, in the dark, for 30 min.
- Pellet by centrifugation, remove the supernatant, resuspend in 1 mL of PBS, and repellet.
- Resuspend in 1 mL of PBS (e.g. 1 x 108 bacteria/mL). Store at 4°C, wrapped in foil, for up to 1 week.
- Bacteria are typically added to phagocytosis assays at a rate of 10-30/macrophage, or 10,000-30,000 per mm2 (Table 2).
- Add desired number of bacteria into the dish/well.
- Mix gently with a pipette to distribute the bacteria evenly.
- If synchronized phagocytosis is required, spin at ~250 x g, 1 min, in the centrifuge.
- Proceed with assay as required.
Bacteria | Stock | CFU/mL/OD600 |
---|---|---|
DH5α | -- | 2.7 x 109 CFU/mL/OD600 |
BL21 | -- | 8.0 x 108 CFU/mL/OD600 |
RFP | RC-105 | 1.6 x 108 CFU/mL/OD600 |
GFP | RC-108 | * |
GFP+RFP | RC-110 | 4.4 x 108 CFU/mL/OD600 |
RFP+smURF | RC-094 | * |
*Not yet determined
Chamber | Well diameter/side | Well area | Low (10k/mm2) | High (30k/mm2) |
---|---|---|---|---|
12-well plate | 18 mm (coverslip) | ~250 mm2 | 2.5 x 106 | 7.5 x 106 |
35 mm dish | 34.4 mm (dish) | 930 mm2 | 9.3 x 106 | 2.8 x 107 |
µ-Slide 18 Well | 5.7 x 6.1 mm | 35 mm2 | 350,000 | 1.0 x 106 |
21-well chamber | 5 mm | 20 mm2 | 200,000 | 600,000 |
32-well chamber | 4.5 mm | 16 mm2 | 160,000 | 480,000 |
36-well chamber | 4.0 mm | 12.6 mm2 | 126,000 | 378,000 |
Old Protocol
- Measure out 4 mL of LB media and add kanamycin to a final concentration of 50 µg/mL.
- Inoculate with the Bacteria Digest 1 (Clone RC-094) bacteria, and grow overnight shaking at 37°C.
- The next morning prepare 40 mL of LB media with 50 µg/mL kanamycin, and inoculate using the whole 4 mL overnight culture prepared in step 2. Incubate, shaking at 37°C, for 1.5 hours.
- At the 1.5 hour mark, add 1 mM IPTG and 25 µM biliverdin. Return to the shaker and incubate at 37°C, for 4 hours.
- At 4 hours remove the flask from the shaker. Prepare a serial dilution, and prepare spread plates on LB-Kan of the 10-9, 10-10, 10-11, 10-12
- Recover the cells by centrifugation, wash once with PBS, and then store in the fridge in 1.5 mL PBS + 25 µM biliverdin.
- The next day use the serial dilution to calculate the CFU/mL in the refrigerator stock. Dilute the stock to 10 x 107/mL in PBS + 20% glycerol, aliquot into PCR tubes (200 µL/tube) and freeze at -80°C until needed.
To use Digestion-Tracking Bacteria in a phagocytosis assay:
- Split MH-S cells onto coverslips (250,000 cells/well), and let recover overnight in 37°C incubator.
- The next morning, discard the media in the wells and replace with 400 μL fresh RPMI media, with 6 μL antibiotic antimycotic solution (10,000 IU/mL penicillin, 10,000 μg/mL streptomycin, 25 μg/mL amphotericin B)
- Thaw 1 tube of Digest Tracking bacteria and add entire tube to a 1.5 mL centrifuge tube. Fill the rest of the tube’s volume with PBS.
- Centrifuge tube at 21,100 g for 3 minutes.
- Carefully remove supernatant and resuspend pellet in 200 μL imaging buffer.
- Add entire tube to coverslip, and live cell image.