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.  
# 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.  
# 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<sup>-9</sup>, 10<sup>-10</sup>, 10<sup>-11</sup>, 10<sup>-12</sup>
# 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 10<sup>7</sup>/mL in PBS + 20% glycerol, aliquot into PCR tubes (200 µL/tube) and freeze at -80°C until needed.


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.
To use Digestion-Tracking Bacteria in a phagocytosis assay:


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
# 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)  
6. 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 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.  
 
# Centrifuge tube at 21,100 g for 3 minutes.  
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.
# Carefully remove supernatant and resuspend pellet in 200 μL imaging buffer.  
 
# Add entire tube to coverslip, and live cell image.
 
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.

Revision as of 20:59, 24 March 2021

  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. 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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