Monack Lab
Protocols

 

Franciscella Infection Protocol

  1. If the bacteria are streaked on a plate (we use Trypic Soy Agar (Becton Dickinson, Sparks, MD) supplemented with 0.1% cysteine (Sigma, St. Louis, MO)), then pick a few colonies with a sterile loop and grow them overnight with aeration in Tryptic Soy Broth (Difco Laboratories, Detroit, MI.) supplemented with 0.2% cysteine (we have a separate bottle of cysteine and dilute it into the autoclaved TSB right before inoculating with bacteria). If you want you can start your overnight from a frozen glycerol stock by scraping a small bit of frozen bacteria and inoculating into the same broth. The important thing is that we use fresh plates i.e. avoid using bacteria that have been sitting on a plate for more than a day.
  2. The next day, dilute the overnight culture 1 to 100 in same broth and grow for 3 hours, with aeration.
  3. Read the OD600 in a spectrophotometer. We use the following conversion OD600 1.0 = 3 x 109 CFU/ml to calculate how much of the culture to use for inoculations. But we also plate dilutions to be sure of our moi's. We make the dilutions of bacteria in fresh, pre-warmed tissue culture media for infecting monolayers.
  4. Francisella is not very efficient at entering nonphagocytic cells. So, you will have to infect with pretty high moi's to get many inside. What I always do is infect with mutliple moi's and look for a correlation with numbers of bacteria and inflammasome activation. For example, I would try 500:1, 200:1, 100:1, 50:1 and measure IL-1beta for all to see if I get decreasing levels of release. Note: if you grow your tissue culture cells in the presence of antibiotics, you will need to wash 3 times with fresh tissue culture media to get rid of the antibiotics.
  5. We centrifuge at 730 x g for 15 minutes to synchronize the infection and then put in the CO2 incubator.
  6. After the bacteria have been given a chance to invade the cells for 30 minutes, add high levels of gentamicin (100 µg/ml) to kill extracellular bacterial. Leave this on for 90 minutes in the CO2 incubator.
  7. Suck off high gent media, wash 3x with warm media and replace with pre-warmed media containing lower gentamicin levels (10 µg/ml).
  8. Let the infection go for varying amounts of time and measure IL-1beta release. Typically, we start looking at about 4 h after infection, but this of course varies depending on moi and cells. Since this would be the first time you are infecting 293 cells, I would suggest measuring the levels of intracellular bacteria by lysing the monolayers with 1% saponin in water for 5 minutes at the various time points and plating dilutions in phosphate-buffered saline to count the numbers of intracellular CFU. You could also look by microscopy, if you decide that you want to do this, we can help by staining for bacteria.

 

RNA Isolation SDS-hot phenol method

Reagents needed:

QIAGEN: Rneasy mini kit, DNAse kit, RNAlater reagent (Ambion Cat# 7021 500ml)
ß-ME, Acid-Phenol:Chloroform, 5:1, pH 4.5 (premixed with IAA- Ambion Cat# 9722), Phenol/Chlorof/IAA 25:24:1, Chlorof/IAA 24:1; DNA-free (Ambion cat #1906)

Starting from liquid culture:

 

Preparation of Bone-marrow-derived macrophages

All reagents have to be endotoxin tested and endotoxin-free!!!

Collection of bone-marrow cells

Collection of macrophages

Thawing cells and experiments


BM Mac media

50 ml FCS (heat-inactivated) 10%
25 ml Horse serum (heat-inactivated) 5% (Gibco or Biochrom)
100 ml L929 cell conditioned supernatant (this varies depending on the potency of the sup.)
5 ml Hepes 1%
5 ml Na-pyruvate 1%
5 ml L-glutamine 1%
fill to 500 ml with DMEM and filter. Use DMEM from Firma Biochrom (#F 0435)

I don’t add antibiotics to the cells. This is so any contamination will grow out, and it will be obvious that the cells are infected, and you can throw them away. I want to avoid a situation where there is a low level contamination and LPS, bacterial lipoproteins, etc. are produced, which would change the activation state of the cells.


DMEM-VM

50 ml FCS (heat-inactivated) 10%
5 ml Hepes 1%
5 ml Na-pyruvate 1%
5 ml L-glutamine 1%
fill to 500 ml with DMEM and filter.


L929 supernatant

Grow the L929 cells in DMEM-VM at 37C, CO2.
108 cells/ triple flask. 200 ml DMEM-VM per triple flask.
Leave for 2-3 days.
12 days later the L929 supernatant is ready. The media will be light orange or yellow, and some cells will start to float.
Collect the supernatant and centrifuge at 1500 rpm for 5 min.
Store aliquots at –80oC. Make aliquots of 50 ml in Falcon tubes, the supernatant should last for at least 1 year.
You have to check the activity of each batch of L929 sup, activity can vary. You can grow 3-5 liters at a time, using about 20 triple flasks.

 

Peritoneal Mouse Macrophage Isolation

  1. Sacrifice mice. Cut peritoneal cavity and pull back skin. Pull membrane away from mouse body and carefully insert needle (try 18 to 20 gauge) with 3mL syringe into peritoneal cavity. Slowly inject cold HBSS (w/o Ca++ and Mg++) + 10% FBS + 1x Pen/Strep. Massage peritoneal cavity with moist gloves for about 1 min. Remove fluid from cavity. Repeat, using same hole for needle. Keep cells on ice during harvesting.
  2. Centrifuge at 2K RPM, 5-10 min at RT
  3. Pour off sup. Respend ~ 2 mice in 10 mLs if doing step 4.
  4. Optional - Slowly overlay onto Ficoll-Paque or Histopaque. Centrifuge at 1K RPM (400g) 10-15 min, RT. (We put 5 mL cell suspension over 5 mL Histopaque in 15 mL conicals)
    Use 5mL plastic pipet to remove interface. Pool in 50 mL conical. Add 40mL media and resuspend with 10mL pipet. Spin 10 min 1K RPM (400g).
  5. Pour off sup. (If you did not do the gradient, just spin them). Add 25 mL media. Resuspend with 10mL pipet. Spin 10 min 1K RPM (400g).
  6. Resuspend cells in residual media (<2 mL – measure this volume). Dilute 5µL into 195µL and count on hemocytometer; assume around 95% of what you counted are macrophages. Expect 5 x 106 cells per mouse (don’t know what to expect for resident peritoneal macs).
  7. Resuspend cells in appropriate volume of media with DMEM + 10% FBS + 1x Pen/Strep + glutamine + Na++pyruvate. Seed appropriately - note these cells cannot be scraped and re-seeded. For example, seed 105 per 96-well well.
  8. Let sit for 2 hrs, wash 2x to remove non-adherents
  9. Incubate overnight and then use.

 

Macrophage activation protocol

RAW 264.7 macrophages (ATCC) were maintained in DMEM (Gibco) containing 10% fetal bovine serum (FBS). 2×105 Cells were seeded per well in 24 well dishes and allowed to adhere overnight. In order to activate cells, media was removed and fresh media containing 100 U/mL recombinant IFN-γ (US Biologicals) and 50 ng/mL LPS (Sigma) were added. Cells were maintained in IFN-γ and LPS-containing medium for 18-24 hrs before infection. Cells that were not activated before infection were infected the day after seeding into 24-well dishes.

 

TUNEL reaction on paraffin tissue sections

  1. place sections which are on glass slides in 60°C oven for 30 min
  2. dewax in xylene bath, 2x 5 min
  3. 96% ethanol 2x 3 min
  4. 90% ethanol
  5. 80% ethanol
  6. ddwater (DDW) rinse
  7. nuclei of tissue sections stripped from proteins by incubating with 20 µg/ml proteinase K in 10 mM trisHCL, pH 7.4-8.0 for 15 in @ RT.
  8. wash in DDW for 2 min, 4 times
  9. rinse 2x in PBS
  10. dry area around tissue section and draw around tissue with PAP pen
  11. overlay tissue section with TUNEL reaction mixture (I use Roche Fluorescein Kit cat#1689795).
  12. incubate in humidified chamber at 37°C for 1 hour.
  13. rinse 3x with PBS
  14. can now stain with antibodies (i.e. rabbit anti-Salmonella primary) for 1 h.
  15. rinse 3x with PBS
  16. stain with secondary antibody (i.e. anti-rabbit-alexa594) for 1 h.
  17. rinse 3x with PBS
  18. overlay with anti-quench (i.e. Vectashield)
  19. place coverslip over sample and seal edges with nail polish.


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