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From 2010.igem.org

Kit Components

• Component A, DMAO: 2x100 µL, 5 mM in DMSO
• Component B, EthD-III: 2x150 µL, 2 mM in DMSO

Preparation of Live and Dead Bacterial Suspensions as Controls

1. Grow 4 mL cultures of your bacteria to late log phase in nutrient broth.
2. Prepare two tubes of 1 mL of the bacteria culture in Eppendorf tubes and centrifuge at 10,000 × g for 10–15 minutes.
3. Remove the supernatant and resuspend the pellet of one tube in 0.3 mL of 0.85% NaCl solution and another tube in 1 mL of 0.85% NaCl.
4. Add 0.7 mL isopropyl alcohol into the tube with 0.3 mL of 0.85% NaCl and mix well (final concentration of isopropyl alcohol: 70%) for preparing dead bacteria.
5. Incubate both samples at room temperature for 1 hour, mixing every 15 minutes.
6. Pellet both samples by centrifugation at 10,000 × g for 10–15 minutes.
7. Resuspend the pellets in 1 mL of 0.85% NaCl and centrifuge again as in step 1.6.
8. Determine the optical density at 670 nm (OD670) for a 3 mL aliquot of the bacterial suspensions in glass or acrylic absorption cuvettes (1 cm pathlength).
9. Use live and dead bacteria at your desired concentration for staining experiments shown below.

Staining Bacteria in Suspension

1. Combine one volume of Component A and two volumes of Component B in a microcentrifuge tube, mix thoroughly and add 8 volumes of 0.85% NaCl solution to derive 100X dye solution.
2. For each 100 uL of your bacteria sample and live and dead bacteria control suspensions, add 1 uL of the dye mixture.
3. Mix thoroughly and incubate at room temperature in the dark for 15 minutes.
4. Trap 5 µL of the stained bacterial suspension between a slide and an 18 mm square coverslip.
5. Observe under a fluorescence microscope equipped with any of the filter sets as below.

Selection of Optical Filters
Longpass and dual emission filters useful for simultaneous viewing of DMAO and EthD-III stains
Omega Filters: XF25, XF26, XF115
Chroma Filters: 11001, 41012, 71010
Bandpass filters for viewing DMAO alone
Omega Filters: XF22, XF23
Chroma Filters: 31001, 41001
Bandpass filters for viewing EthD-III alone
Omega Filters: XF32, XF43, XF102, XF108
Chroma Filters: 31002, 31004, 41002, 41004

Flow Cytometry

1. Adjust the E. coli suspensions (live and killed) to 1 × 108 bacteria/mL (~0.03 OD670), then dilute them 1:100 in filter-sterilized dH2O to reach a final density of 1 × 106 bacteria/mL if needed.
2. Mix 11 different proportions of E. coli in 16 × 125 mm borosilicate glass tubes according to Table 1. The volume of each of the 11 samples will be 1 mL.
3. Mix 12 µL of Component A with 24 µL of Component B in a microcentrifuge tube. Add 3 µL of the combined reagent mixture to each of the 11 samples and mix thoroughly by pipetting up and down several times.
   Note: It may be desirable to prepare additional bacterial samples for staining with component A alone (stain both live and dead bacteria ) and with Component B alone (stain dead bacteria only).
4. Incubate at room temperature in the dark for 15 minutes.
5. Analyze each bacterial sample by flow cytometry using the setting for fluorescein for DMAO positive cells and propidium iodide for EtD-III positive cells.

Table 1: Volume of Live- and dead-cell suspension to mix to achieve desired ratio of live:dead cell population

Ratio of Live:Dead Cells mL Live-Cell	Suspension mL Dead-Cell	Suspension
0:100	0	1.0
10:90	0.1	0.9
20:80	0.2	0.8
30:70	0.3	0.7
40:60	0.4	0.6
50:50	0.5	0.5
60:40	0.6	0.4
70:30	0.7	0.3
80:20	0.8	0.2
90:10	0.9	0.1
100:0	1.0	0

Adapted from: http://www.openwetware.org/images/b/b9/Cell_Death_Assay.pdf