NPN Uptake Assay / Flourescence Measurements

Categorized: Outer Membranes

References:

  • Hancock & Wong. AAC. Vol.26, No.1:48 – 52. July, 1984.
  • Loh et. al. AAC. Vol. 26, No. 4:546 – 551. October, 1984.
  • Hancock et. al. AAC. Vol. 35, No. 7:1309 – 1314. July, 1991.

Materials:

Buffer:

  • 5 mM sodium Hepes pH7.2
  • 5 uM CCCP (better) or 5mM KCN
  • 5 mM glucose

Note: Filter sterilize buffer if desired but DO NOT AUTOCLAVE any of its components except dH2O. If using CCCP (which is unstable and light sensitive) make only enough working solution for that day. Solubilize stock solution in EtOH and store at -20oC in the dark.

1-N-phenylnapthylamine (NPN ):

  • Make 5 mM stock solution = 10.95 mg in 10 ml acetone. Dilute 1/10 to make a 0.5 mM working solution. Store both stock and working solutions at -20oC between experiments.

NaCl & MgCl2 (may not need):

  • Make serial dilutions over the range you wish to test. MgCl2: make 300 mM, 100 mM, 30 mM & 10 mM (use 1/100). NaCl: make 3 M, 1.5 M, .75 M (use 1/10)

Antibiotics:

  • Make up solutions in buffer or distilled water. Make serial dilutions 100X the desired final concentration so you are always adding a constant volume to the cuvette,
  • i.e. Polymyxin B;
  • adding 10 µl to a 1 ml cell solution
  • make 0.64 mg/ml –> final concentration 6.4 µg/ml
  • dilute 1/2: 0.32 mg/ml –> final concentration 3.2 µg/ml
  • dilute 1/2: 0.16 mg/ml –> final concentration 1.6 µg/ml
  • Each antibiotic is used over a different range
Polymyxin B (PxB) 0.1 µg/ml – 6.4 µg/ml
Gentamicin 1 µg/ml – 100 µg/ml
Erythromycin 500 µg/ml – 2000 µg/ml

Preparing Cells:

A. Use 1 ml of overnight culture to innoculate 50 mls of media. Incubate 37oC, shaking. Grow to OD600 = 0.4 – 0.6.B. Spin down cells: Sorvall SS34, Room Temp., 10,000 rpm, 5 min. or Silencer, Room Temp., 3500 rpm, 10 min.

C. Wash 1X in buffer. (Turn on fluorescent spec. now)

D. Resuspend in buffer to OD600 = 0.5. Record OD600.

Note: Don’t grow up large batches of cells; they are not stable enough to assay all day. It is better to grow another batch while you are assaying the first ones if you need a lot of cells. This avoids getting leaky cells which results in high background fluorescence of NPN only

Note: For Pseudomonas: keep at room temp. DO NOT SPIN AT 4ºC. Use the silencer in room 235A if the Sorvall is too cold. (This also helps prevent leaky cells.)

Using the Fluorescent Spectrophotometer (Perkin-Elmer 650 – 105):

  1. ON POWER SUPPLY: Switch power to on. Count to 10. Press Lamp button. Check if lamp is on by holding white piece of paper in front of the beam inside the spec. Make sure the side ‘door’ is pushed in (therefore open).
  2. Set excitation wave length to 350. Set emission wave length to 420. Set both slit widths to 5. sett the range to 1. Turn the spec. on, the chart recorder on and switch the multimeter to “DCV2”.

Check the other settings which should be:

PM GAIN ‘NORM’
O SUPP ‘ON’ (May need to adjust later)
RESPONSE ‘NORM’
MODE ‘NORM’
SCAN ‘STOP’
SCAN SPEED (IRRELEVANT)

To “Standardize Assay”:

  1. Add 1.0 ml cell solution to cuvette. Place inside spec.. Open the side door (i.e. push in). Using the zero,adjust and zero suppression on spec. (adjust the knobs till the multimeter reads 0.0) Now adjust with the ‘zero’ knob on the chart recorder till the pen is at the baseline (or + 5 – 10% if you prefer)
  2. Add 20 ul of 0.5 mM NPN (final conc. = 10 uM). Top cuvette with parafilm and shake. Place in spec.; measure for 2 – 5 seconds.
  3. Remove cuvette and add 10 µl of 0.64 mg/ml PxB. Shake; place in spec. Using the range and sensitivity knobs, adjust until the pen is at 90% full deflection.
  4. Repeat steps 1 – 3 until you have PxB 6.4 µg/ml giving 90% deflection and you have zeroed properly after changing the range &/or sensitivity.

Now you are ready to start! Try adding solutions and returning the cuvette quickly.

The Assay:

  1. Add 1 ml of cells (OD600 = 0.5) to cuvette. Measure 2 – 5 seconds.
  2. Add 20 ul NPN 0.5 mM (shake to mix). Measure 2 – 5 seconds.
  3. Add 10 ul antibiotic 100X desired final concentration (shake to mix). Measure till max. level reached (1 – 5 min.). If testing effect of NaCl or MgCl2, add each in between NPN and antibiotic.
  4. Don’t assay more than 2 hours – the cells don’t work as well as time goes on.

Be sure to assay controls:

  1. cells only
  2. cells + NPN only (no antibiotic)
  3. cells + NPN + antibiotic solvent
  4. NPN in buffer only (no cells, no antibiotic)
  5. NPN in buffer only + antibiotic (no cells)