TITLE: Nanoluciferase Assay in C. elegans
AUTHORS: Emily Troemel, Ivana Sfarcic, Theresa B, Erin C Daniels

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>A highly sensitive Nanoluciferase (NanoLuc)-based method in multi-well format to detect constitutive and inducible gene expression in whole </span><span style = "font-style:italic;">C. elegans</span><span>. Worms have to be lysed for this end-point assay, no spatial resolution of expression can be detected with this protocol.</span></div></div>

[STEPS]
?. A.Grow up/Treat Worms and Prepare Lysate

Grow up and/or treat worms as required, usually on 3.5 or 6 cm NGM plates seeded with OP50-1. N2 can be included as no-Nanoluciferase control, ERT513 (unc-119 (ed3) III; jySi35 [pET618 (vha-6p::NANOLUC::unc-54 3' UTR, unc-119 (+)])or ERT529 (unc-119 (ed3) III; jySi40 [pET635 (vha-6p::NANOLUC::3XFLAG::unc-54 3' UTR, unc-119 (+)]) can be used as Nanoluciferase (NanoLuc) expressing control. Well-fed adults give highest signal.Prepare 1.7 ml microfuge tubes with 80 - 100 ul 1x Lysis BufferPick worms into Lysis Buffer, making sure that they were released from the (eyelash) pickAdd approximately 5 - 10 Silicon Carbide beads to tube with wormsPlace Disruptor Genie to 4°C and vortex samples for 4 minutesSpin down sample for 1 minute at max speed with a table top centrifuge
Note*: Possible to freeze supernatant (worm lysate) to -80°C for later use. Transfer supernatant to new 1.7 ml tube and freeze.Transfer 50 ul of the supernatant (worm lysate) to well of white 96-well platePrepare 25 ul of NanoGlo Reagent per sample well (1 part NanoGlo Substrate + 50 parts NanoGlo Buffer)
Note: Do not resuspend worm lysate and NanoGlo Reagent by pipetting up and down, bubbles might mess up reading. Shaking on plate shaker is sufficient to mix.Add 25 ul of NanoGlo Reagent to every well with worm lysate (using repeater pipette is most sufficient to stay bubble free)Agitate plate for 10 minutes at room temperature.ii.Prepare Lysate of ≥ 50 to < 1,000 Worms

Wash worms from growth plate into 1.7 ml tubes with M9-T (M9 with 0.1 % Tween-20)Spin worms down at 2,000xg for 1 minute, remove supernatant to 100 ul lineAdd 300 ul of 1X Lysis BufferSpin worms down at 2,000xg for 1 minute, remove supernatant to 100 ul lineAdd approximately 10-15 Silicon Carbide beads to tube with wormsPlace Disruptor Genie to 4°C and vortex samples for 4 minutesSpin down sample for 1 minute at max speed with a table top centrifuge
Note*: Possible to freeze supernatant (worm lysate) to -80°C for later use. Transfer supernatant to new 1.7 ml tube and freeze.Transfer 50 ul of the supernatant (worm lysate) to well of white 96-well platePrepare 25 ul of NanoGlo Reagent per sample well (1 part NanoGlo Substrate + 50 parts NanoGlo Buffer)
Note: Do not resuspend worm lysate and NanoGlo Reagent by pipetting up and down, bubbles might mess up reading. Shaking on plate shaker is sufficient to mix.Add 25 ul of NanoGlo Reagent to every well with worm lysate (using repeater pipette is most sufficient to stay bubble free)Agitate plate for 10 minutes at room temperature.iii.Prepare Lysate of ≥ 1,000 Worms

Wash worms from growth plate into 15 ml tubes with M9-T (M9 with 0.1 % Tween-20)Spin worms down at 2,000xg for 1 minute, remove supernatant to 300 ul lineTransfer worms to 1.7 ml tubes with glass Pasteur pipetteAdd 300 ul of 1X Lysis BufferSpin worms down at 2,000xg for 1 minute, remove supernatant to 100 ul line or above in case worm pallet is largerAdd approximately 15-20 Silicon Carbide beads to tube with wormsPlace Disruptor Genie to 4°C and vortex samples for 4 minutesSpin down sample for 1 minute at max speed with a table top centrifuge
Note*: Possible to freeze supernatant (worm lysate) to -80°C for later use. Transfer supernatant to new 1.7 ml tube and freeze.Transfer 50 ul of the supernatant (worm lysate) to well of white 96-well platePrepare 50 ul of NanoGlo Reagent per sample well (1 part NanoGlo Substrate + 50 parts NanoGlo Buffer)
Note: Do not resuspend worm lysate and NanoGlo Reagent by pipetting up and down, bubbles might mess up reading. Shaking on plate shaker is sufficient to mix.Add 50 ul of NanoGlo Reagent to every well with worm lysate (using repeater pipette is most sufficient to stay bubble free)Agitate plate for 10 minutes at room temperature.Wash worms from growth plate into 1.7 ml tubes with M9-T (M9 with 0.1 % Tween-20)Spin worms down at 2,000xg for 1 minute, remove supernatant to 100 ul lineAdd 300 ul of 1X Lysis BufferSpin worms down at 2,000xg for 1 minute, remove supernatant to 100 ul lineAdd approximately 10-15 Silicon Carbide beads to tube with wormsPlace Disruptor Genie to 4°C and vortex samples for 4 minutesSpin down sample for 1 minute at max speed with a table top centrifuge
Note*: Possible to freeze supernatant (worm lysate) to -80°C for later use. Transfer supernatant to new 1.7 ml tube and freeze.Transfer 50 ul of the supernatant (worm lysate) to well of white 96-well platePrepare 25 ul of NanoGlo Reagent per sample well (1 part NanoGlo Substrate + 50 parts NanoGlo Buffer)
Note: Do not resuspend worm lysate and NanoGlo Reagent by pipetting up and down, bubbles might mess up reading. Shaking on plate shaker is sufficient to mix.Add 25 ul of NanoGlo Reagent to every well with worm lysate (using repeater pipette is most sufficient to stay bubble free)Agitate plate for 10 minutes at room temperature.iii.Prepare Lysate of ≥ 1,000 Worms

Wash worms from growth plate into 15 ml tubes with M9-T (M9 with 0.1 % Tween-20)Spin worms down at 2,000xg for 1 minute, remove supernatant to 300 ul lineTransfer worms to 1.7 ml tubes with glass Pasteur pipetteAdd 300 ul of 1X Lysis BufferSpin worms down at 2,000xg for 1 minute, remove supernatant to 100 ul line or above in case worm pallet is largerAdd approximately 15-20 Silicon Carbide beads to tube with wormsPlace Disruptor Genie to 4°C and vortex samples for 4 minutesSpin down sample for 1 minute at max speed with a table top centrifuge
Note*: Possible to freeze supernatant (worm lysate) to -80°C for later use. Transfer supernatant to new 1.7 ml tube and freeze.Transfer 50 ul of the supernatant (worm lysate) to well of white 96-well platePrepare 50 ul of NanoGlo Reagent per sample well (1 part NanoGlo Substrate + 50 parts NanoGlo Buffer)
Note: Do not resuspend worm lysate and NanoGlo Reagent by pipetting up and down, bubbles might mess up reading. Shaking on plate shaker is sufficient to mix.Add 50 ul of NanoGlo Reagent to every well with worm lysate (using repeater pipette is most sufficient to stay bubble free)Agitate plate for 10 minutes at room temperature.Wash worms from growth plate into 15 ml tubes with M9-T (M9 with 0.1 % Tween-20)Spin worms down at 2,000xg for 1 minute, remove supernatant to 300 ul lineTransfer worms to 1.7 ml tubes with glass Pasteur pipetteAdd 300 ul of 1X Lysis BufferSpin worms down at 2,000xg for 1 minute, remove supernatant to 100 ul line or above in case worm pallet is largerAdd approximately 15-20 Silicon Carbide beads to tube with wormsPlace Disruptor Genie to 4°C and vortex samples for 4 minutesSpin down sample for 1 minute at max speed with a table top centrifuge
Note*: Possible to freeze supernatant (worm lysate) to -80°C for later use. Transfer supernatant to new 1.7 ml tube and freeze.Transfer 50 ul of the supernatant (worm lysate) to well of white 96-well platePrepare 50 ul of NanoGlo Reagent per sample well (1 part NanoGlo Substrate + 50 parts NanoGlo Buffer)
Note: Do not resuspend worm lysate and NanoGlo Reagent by pipetting up and down, bubbles might mess up reading. Shaking on plate shaker is sufficient to mix.Add 50 ul of NanoGlo Reagent to every well with worm lysate (using repeater pipette is most sufficient to stay bubble free)Agitate plate for 10 minutes at room temperature.
?. B.Measure Luminescent Signal with NovoStar Plate Reader

Use at least two wells with 1X Lysis Buffer with NanoGlo Reagent as blank (ideally, Lysis Buffer was vortexed with Silicon Carbide beads)Switch on plate reader (switch located at right site of reader), open black sliding door on top of plate reader; Install optic fiber for luminescent measurement for top reading, close sliding door tightlyOpen “NovoStar” softwareChoose “Setup” > “Reader configuration” > “Luminescence”Choose “Test setup” > “Test protocol” > “Bright Glo” (new window opens) > “Edit” > “Layout” > mark samples (and blank) according to your plate design (only those wells are measured)
Bright Glo measures signal for 1 second per well without filters.Choose “Measure” > “Plate out” > insert uncovered plate > “Plate in”Choose “Measure” (new window opens) > “Measure”
Note: Before pressing “Measure” in new window, shaking and optic settings can be adjusted:
 7.i) Shaking settings should be linear shaking 1mm: additional shaking time: 10s before plate reading; positioning delay 0.2 sec. 
 7.ii) Gain should be adjusted on well with highest expected signal. If that’s unknown, click plate number “96” in upper left corner to determine best well for gain adjustment. Highest possible gain is 4095.To open results, choose “Results” > “Open last test Run” (MARS software opens and displays results in plate or table format as well as a summary of program settings)Export data from each tab with Excel buttonTo shut down, close MARS software, remove plate and close NovoStar software, shut down computer, switch off NovoStar plate reader