Preparation of Digestion-Tracking Bacteria

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:

1. Prepare ~2 mL of LB + the appropriate antibiotic(s) at a 1X concentration in a 14 mL snap-cap tube.
2. Inoculate with a loop of the desired bacteria from the frozen stock.
3. Grow overnight, 37°C, shaking.
4. Prepare 2 mL of fresh LB + sufficient antibiotics for 2.5 mL of medium in a 14 mL snap-cap tube.
5. Inoculate this new tube with 400 µL of the overnight culture.
6. Grow for 1 hr, 37°C, shaking.
7. Add 2.5 µL IPTG to the culture.
   • If using a smURF-containing bacteria, add 2.5 µL (25 µM) biliverdin at this time.
8. Incubate overnight, 37°C, shaking.
9. 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).
10. 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.
11. 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.
12. Pellet by centrifugation, remove the supernatant, resuspend in 1 mL of PBS, and repellet.
13. 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.
14. 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.

Table 1: CFU/OD600 of Various Bacterial Strains

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

Table 2: Common Bacterial Additions for Phagocytosis Assays

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

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Old Protocol

1. Measure out 4 mL of LB media and add kanamycin to a final concentration of 50 µg/mL.

1. Inoculate with the Bacteria Digest 1 (Clone RC-094) bacteria, and grow overnight shaking at 37°C.
2. 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.
3. 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.
4. 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
5. Recover the cells by centrifugation, wash once with PBS, and then store in the fridge in 1.5 mL PBS + 25 µM biliverdin.
6. 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:

1. Split MH-S cells onto coverslips (250,000 cells/well), and let recover overnight in 37°C incubator.
2. 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)
3. 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.
4. Centrifuge tube at 21,100 g for 3 minutes.
5. Carefully remove supernatant and resuspend pellet in 200 μL imaging buffer.
6. Add entire tube to coverslip, and live cell image.