1. Count cells and prepare a mastermix to have 80’000 cells or 600’000 cells per well for adherent and suspension cells, respectively. Prepare enough mastermix for all desired controls, concentrations, incubation periods, and replicates.
2. Distribute the cells into the wells of a 96well plate, 200 µL per well.
3. Adherent cells must be allowed to settle for 2 hours previous to the start of the experiment.
4. Apply 5 µL of the desired dillutions of sTRAIL to the appropriate wells and incubate for 2 to 6 hours at 30ºC.
5. After incubation, separate cells from their medium (spin down suspension cells, remove medium from wells with adhesive cell.
6. Wash 3 times with DPBS.

For detection of apoptosis, Annexin V-Alexa488 can be used. It will bind to externalizes phosphatidylserine on apoptotic cells.

7. Resuspend cells in Annexin binding buffer (link!) and add 5 µL per sample. Incubate at RT for 15 min.
8. Dillute Annexin V-Alexa488 by addition of 50 µL of Annexin binding buffer. (link!) Alternatively, engineered E. coli bacteria expressing our INP-Annexin V construct along with RFP (link to biobrick annexin and composite PL5) can be used for detection.
9. Analyse cells by FACS.
10. PI staining can be used to distinguish apoptotic from necrotic cells. Apply 5 µL of PI to the respective samples 5 min prior to FACS analysis.

Application of the appropriate positive controls (eg. PMA for Jurkat cells) and control staining (PI for necrotic cells) will lead to more detailed and reliable results.

Alternative induction of apoptosis using PMA

Susceptible cell lines can be incubated with PMA in appropriate incubation period and concentrations as a positive control to sTRAIL induced apoptosis. The following protocol is designed for Jurkat cells.

1. Incubate cells with blabla PMA and Ionomycin for 24 hours.
3. Wash cells 3 times with DPBS and resuspend in Annexin V binding buffer for detection with Annexin V-Alexa488.

1. Count cells and prepare a mastermix to have 80’000 cells or 600’000 cells per well for adherent and suspension cells, respectively. Prepare enough mastermix for all desired time points and replicates. Counting must be very accurate if the determination of the lactate output per cell is the aim of the experiment. Consider using a cell counter.
2. Distribute the cells into the wells of a 24well plate, 500 µL per well.
3. Adherent cells must be allowed to settle for 2 hours previous to the start of the experiment.
4. At the beginning of the measurement, the medium has to be exchanged to start at the minimal level. To this end, spin down the cells at 250 rcf for 3 min and resuspend the pellet in 500 µL of fresh, prewarmed medium. The sample for time zero has to be taken right after the exchange of the medium.
5. Samples are collected in regular time intervals by transferring the medium into an Eppendorf tube. Centrifuge samples at 250 rcf for 3 min and transfer the cell free medium into a new Eppendorf tube.
6. Samples can be stored at 4 to 8 ºC for analysis at a later time point.
7. The lactate concentration of the collected samples can be determined using the L-Lactate Kit according to its instructions. In order to get good results with this kit, it is essential to use medium free of serum and phenol red.

1. Dillute overnight cultures (biological triplicates) to an OD600 of 0.5 in a final volume of 20 µL.
2. Add primary antibody to a final concentration of 5 µg/mL and incubate for 2 h at 37 ºC.
3. Centrifuge at low speed for 2 min to precipitate cells. Discard supernatant to remove excess primary antibody.
4. Resuspend cells in LB medium including antibiotics and add secondary antibody to a concentration of 1 µg/mL and incubate for 1 h at 37 ºC.
5. Centrifuge at low speed for 2 min to precipitate cells. Discard supernatant to remove excess secondary antibody.
6. Resuspend cells in PBSand observe fluorescence by FACS or Confocal microscopy.

1. Wash beads twice in PBS.
2. Dilute stock 6x and use 25uL per two experiments.
3. Incubate 1 µg anti-Annexin antibody on beads for 30 min on ice.
4. Wash beads twice in PBS.
5. Incubate beads with bacteria expressing Annexin V on their surface for 30-60 min on ice. As a control Annexin V-Alexa488 can be used.
use a lot of bacteria
6. Wash beads in PBS at least twice.
7. Analyze beads by FACS or microscopy.

1. Wash beads twice in PBS.
2. Use 5uL beads suspension per experiment and incubate them with biotinylated phosphatidylserine for 30min at RT.
3. Wash beads twice in PBS and once in Annexin binding buffer.
4. Incubate beads with bacteria expressing Annexin V on their surface for 30-60 min. Use 25uL of a bacterial preculture. To support binding of Annexin V to phosphatidylserine, add 25uL of Annexin binding buffer. As a control beads without PS coating, as well as bacteria without the INP-Annexin V construct can be used.
5. Wash beads in PBS three times.
6. Analyze beads by FACS or microscopy.

Western Blot

1. Make overnight cultures of all samples and controls.
2. Make quick extract of overnight cultures at OD600=2.0 by spinning them down and re- disolving the pellet in 50 µL of 1x SDS loading buffer and boiling it for 10 min. Then spin down at max speed for 10 min.
3. Take 8 mL of the supernatant and load onto SDS-PAGE gel.
4. Run according to common sense.
5. Equilibrate gel in transfer buffer for 15 min at RT. Pre-wet 2 fibre pads, 2 pieces of filter paper of appropriate size, and 1 piece of nitrocellulose in transfer buffer.
6. Set up transfer sandwich on the black side of the cassette, in a plastic tray, in the order filter pad, filter paper, nitrocellulose, gel, filter paper, fibre pad, black side of cassette. Add buffer between each layer and avoid air bubbles! Fold the white half of the cassette over the sandwich and close.
7. Insert cassette into the transfer apparatus in the correct orientation.
8. Insert ice block (filled with ice or with water), fill box with transfer buffer.
9. Transfer at 250 mA (or 100 V) for 45 minutes.
10. Disassemble the transfer apparatus. Shake the membrane in water for 5 minutes.
11. Block the blot in 10 ml of 5% non-fat dry milk in 1x PBS + 0.15% Tween 20 for 1 hour at RT.
12. Remove block, add primary antibody in 5 ml of block, incubate for 1 hour at RT. Typical concentrations of primary antibodies are 1:5000, 0.2-2 µg/mL.
13. Remove primary antibody and wash 3x with 10 ml PBS, 5 minutes each at RT.
14. Add secondary antibody in appropriate concentration. Incubate for 1 hour at RT.
15. Remove secondary antibody and wash four times with 10 mL PBS, 5 min each at RT.
16. Detect on Li-COR.

Lactate response measurements

1. Dilute overnight culture in new medium plus antibiotics by adding 40 µL of overnight culture to 2.96 mL of medium.
2. Add diluted cultures into 96 well plate.
3. Incubate for 90 to 120 min at 37 ºC.
4. In the meantime prepare 300 µL of desired concentrations of lactate. Careful: prepare 20x stock!
5. Add 10 µL of the lactate solution to the samples in the 96 well plate.
6. Set up kinetic cycle in plate reader:
   1. Shaking for 300 s, orbital, 6 mm amplitude.
   2. Measure OD600.
   3. Shaking for 5 s, orbital, 6 mm amplitude.
   4. Measure fluorescence at excitation at 488 and emission at 530. Set gain manually to 70.
7. Run kinetic cycle for 7 hours.

Alternatively, the reaction of bacteria to any sample of interest containing lactate can be determined in this manner. Variation of the nutrient content of the medium used for these experiments may be considered for further characterization.

AHL response experiments

1. Dilute overnight culture in new medium plus antibiotics by adding 40 µL of overnight culture to 2.96 mL of medium.
2. Add diluted cultures into 96 well plate.
3. Incubate for 90 to 120 min at 37ºC.
4. In the meantime prepare 20x stock solutions of desired concentrations of AHL.
5. Add 10 µL of the lactate solution to the samples in the 96 well plate.
6. Set up kinetic cycle in plate reader:
   1. Shaking for 300 s, orbital, 6 mm amplitude.
   2. Measure OD600.
   3. Shaking for 5 s, orbital, 6 mm amplitude.
   4. Measure fluorescence at excitation at 488 and emission at 530. Set gain manually to 70.
7. Run kinetic cycle for 7 hours.

1. protocol

1. Introduce mammalian cells of interest into the wells of a 12well plate. Adherent cells must be allowed to settle for 2 hours previous to any experiment.
2. Add 500 µL of a bacterial preculture to the mammalian cells. For analysis using microscopy it is best to use bacteria expressing a fluorophore.
3. Incubate for 4 hours. Do not incubate in a mammalian cell incubator to avoid contamination of neighbouring cultures!
4. Analyze samples using a microscope. Check for colocalization or possible internalization of bacteria by mammalian cells, as well as viability of mammalian cells with or without bacteria.

1. Apoptosis can be induced in sTRAIL susceptible cells as described elsewhere (link to above) using the optimal incubation time with sTRAIL using the optimal concentration of this agent.
2. Detection of apoptotic cells can be performed by applying xx µL of INP-Annexin V and RFP expressing E. coli to the samples followed by 30 min incubation.
3. Analyse samples by FACS.

1. howto

1. protocol

Cryostock of bacteria

1. Add 1 mL of 40% glycerol to a cryogenic vial.
2. Add 1 mL sample from the bacterial culture.
3. Vortex the vial.
4. Store in -80ºC freezer.

Preparation of chemically competent cells

1. Take a trace of Escherichia coli from a glycerol stock vial and put it on a LB plate with the correspondent antibiotic.
2. Incubate overnight at 37ºC.
3. Pick a colony and inoculate 10mL LB medium containing the relevant antibiotic. Grow overnight at 37ºC.
4. Add 1mL overnight culture to 100mL prewarmed LB medium containing the relevant antibiotic in a 500mL flask, and shake at 37ºC until an OD₆₀₀ of 0.5 is reached (usually 90-120 min).
5. Cool the culture on ice 5min, and transfer the culture to a sterile, round-bottom centrifuge tube.
6. Collect the cells by centrifugation at low speed (5 min, 4000 xg, 4ºC).
7. Resuspend the cells gently in cold (4ºC) TFB1 buffer (30 mL for 100 mL culture) and keep the suspension on ice for 90min.
8. Collect the cells by centrifugation (5min, 4000 xg, 4ºC).
9. Discard the supernatant carefully. Always keep the cells on ice.
10. Resuspend the cells carefully in 4 mL ice-cold TFB2 buffer
11. Prepare aliquots of 100-200µL in sterile microcentrifuge tubes and freeze in dry-ice-ethanol mix. Store the competent cells at -70ºC

Transformation of chemically competent cells

1. Take 1 µL DNA to be transformed and put into a cold sterile 1.5 mL microcentrifuge tube, and keep on ice.
2. Thaw an aliquot of frozen competent cells on ice.
3. Resuspend the cells and transfer 50 of the cell suspension into the microcentrifuge tube with the plasmid DNA, mix carefullu, and keep on ice for 20 min.
4. Transfer the tube to a 42ºC water bath for 90 s.
5. Add 500 µL SOC medium to the cells and incubate for 60-90 min at 37ºC. Shaking increases transformation efficiency.
6. Plate 100 µL on LB-agar plates containing antibiotics. Incubate the plates overnight at 37ºC.

Electrocompetent cells

1. Dillute overnight cultures and let them grow to an OD600 of 0.6 to 0.8. Use approximately 2 mL of culture per transformation.
2. Spin down the cells at half maximal speed for 10 min and remove the supernatant.
3. Wash cells three times in 1 mL ice cold water to remove all residual salts of the LB medium.
4. Resuspend cells in 50 µL ice cold water and add 1 µL of DNA.
5. Pipett the cells into a clean cuvette and apply an electric pulse. Check the voltage. If it is above 5 you are fine.
6. Immediately resuspend the cells in 1mL fresh medium and transfer them to an eppi. Incubate at 37ºC for 1h.
7. Plate 100 µL and 300 µL onto LB plates containing the appropriate antibiotics.

Digest with restriction enzymes

1. Produce the following master mix:
1. Water up to 50µL
2. 1µg of DNA
3. 5µL CutSmart buffer (bought in BioLabs)
4. 1µL of each restriction enzyme
2. Incubate 1 hour at 37ºC.
3. Inactivate the enzyme with the correspondent temperature during 20 minutes.
4. Purify the product using an agarose gel.

Ligation

1. Produce the following master mix on ice:
1. Water up to 20µL
2. 50 ng of vector DNA
3. 37.5 ng of insert DNA
4. 2µL T4 DNA ligase buffer (bought in BioLabs)
2. Incubate overnight at 16ºC (if the ends are sticky).
3. Heat inactivate at 65ºC for 10 minutes.
4. Put on ice and transform.

Gibson Assembly

1. Calculate the concentration needed of the correspondent fragments:
1. For 2 fragments: 2-3x molar excess of the insert.
2. For 2-3 fragments: total molarity 0.02-0.5 pmol.
3. For 4-6 fragments: total molarity 0.2-1 pmol.
2. Put 5 µL of DNA with 15 µL of Gibson Assembly mix. 6mL of this mix consist in:
1. 3 mL of 1M Tris-HCl pH 7.5
2. 150 µ of 2 M MgCl₂
3. 60 µl of 100 mM dGTP
4. 60 µl of 100 mM dATP
5. 60 µl of 100 mM dTTP
6. 60 µl of 100 mM dCTP
7. 300 µL of 1M DTT
8. 1.5 g PEG-800
9. 300 µl of 100 mM NAD
3. Incubate at 50ºC for 60 minutes.
4. Transform the correspondent microorganism.

Colony PCR

1. Take 20 µL of LB and put one colony inside.
2. Prepare master mix for PCR containing:
1. Water up to 25µL
2. 1µL of resuspended cells
3. 10µL ThermoPol buffer (bought in BioLabs)
4. 10 mM dNTPs
5. 10 µM forward primer
6. 10 µM reverse primer
7. 0.125 µL of taq DNA polymerase
3. Use the thermocycler with the following conditions:
1. Initial denaturation at 98ºC for 30 seconds.
2. 35 cycles, with a denaturation at 98ºC for 10 seconds; primer hybridization temperature depending on the primer for 25 seconds; and an elongation of the sequence at 72ºC for 30 seconds per each kb.
3. Final extension at 72º for 10 minutes.

Phusion PCR

1. Prepare master mix for PCR containing:
1. Water up to 50 µL
2. 1 µL of resuspended cells
3. 10 µL Phusion HF buffer (bought in BioLabs)
4. 10 mM dNTPs
5. 10 µM forward primer
6. 10 µM reverse primer
7. 0.5 µL of phusion DNA polymerase
2. Use the thermocycler with the following conditions:
1. Initial denaturation at 98ºC for 30 seconds.
2. 35 cycles, with a denaturation at 98ºC for 10 seconds; primer hybridization temperature depending on the primer for 25 seconds; and an elongation of the sequence at 72ºC for 30 seconds per each kb.
3. Final extension at 72º for 10 minutes

Adherent cell lines

1. Remove the medium by aspirating it using the vacuum pump.
2. Wash cells with 10 mL of DPBS to remove traces of medium.
3. Remove the DPBS by aspirating it using the vacuum pump.
4. Incubate the cells for 2-5 min with Accutase or Trypsin. Use one or two mL for a 25 squarecentimeter flask and 75 squarecentimeter flask, respectively.
5. Check if the cells are detached after the incubation using the microscope or checking by eye. If they are detached resuspend the cells in approximately 7mL of fresh prewarmed medium to neutralize Trypsin or Accutase. Transfer the resuspended cells into a 15 mL falkon tube.
6. Spin down the cells for 3 min at 250 rcf.
7. Remove the medium by aspirating it using the vacuum pump.
8. Resuspend the cells in 3 to 5 mL depending on size of the pellet.
9. Count cells using a Neubauer chamber.
10. Prepare new cultures in 5 or 10 mL in 25 or 75 squarecentimeter flasks, respectively. Seed xxx or xxx cells per squarecentimeter to reach confluency after 2 or 3 days, respectively.

Suspension cells

1. Transfer the whole volume of the culture into a 15 mL falkon tube.
2. Spin down the cells for 3 min at 250 rcf.
3. Remove the medium by aspirating it using the vacuum pump.
4. Resuspend the cells in 3 to 5 mL depending on size of the pellet.
5. Count cells using a Neubauer chamber.
6. Prepare new cultures in 5 or 10 mL in 25 or 75 squarecentimeter flasks, respectively. Seed xxx or xxx cells per squarecentimeter to reach confluency after 2 or 3 days, respectively.