Preparation of Digestion-Tracking Bacteria: Difference between revisions

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1. Measure out 4 mL of LB media and add kanamycin to a final concentration of 50 µg/mL.  
1. Measure out 4 mL of LB media and add kanamycin to a final concentration of 50 µg/mL.  
2. Inoculate with the Bacteria Digest 1 (Clone RC-094) bacteria, and grow overnight shaking at 37°C.  
2. Inoculate with the Bacteria Digest 1 (Clone RC-094) bacteria, and grow overnight shaking at 37°C.  
3. 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. 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.  
4. 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 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.  
5. 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. 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
6. Recover the cells by centrifugation, wash once with PBS, and then store in the fridge in 1.5 mL PBS + 25 µM biliverdin.  
6. Recover the cells by centrifugation, wash once with PBS, and then store in the fridge in 1.5 mL PBS + 25 µM biliverdin.  
7. 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.
7. 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:  
To use Digestion-Tracking Bacteria in a phagocytosis assay:  
1. Split MH-S cells onto coverslips, let recover overnight in 37°C incubator.  
1. Split MH-S cells onto coverslips, 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)  
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. The next morning, 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.  
3. The next morning, 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.  
4. Centrifuge tube at 21,100 g for 3 minutes.  
5. Carefully remove supernatant and resuspend pellet in 200 μL imaging buffer.  
5. Carefully remove supernatant and resuspend pellet in 200 μL imaging buffer.  
6. Add entire tube to coverslip, and live cell image.
6. Add entire tube to coverslip, and live cell image.

Revision as of 20:54, 24 March 2021

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

2. Inoculate with the Bacteria Digest 1 (Clone RC-094) bacteria, and grow overnight shaking at 37°C.

3. 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.

4. 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.

5. 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

6. Recover the cells by centrifugation, wash once with PBS, and then store in the fridge in 1.5 mL PBS + 25 µM biliverdin.

7. 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, 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. The next morning, 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.

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