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# Setup for live cell
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## 1. Turn on the main CO$_2$ supply
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The carbon dioxide is supplied from the tank opposite the Nikon Ti room. When CO$_2$ is not being used the tanks should all be off and the main gauge reading 0 kPa.
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Open the active tank by turning the tap on the top of the tank. The main gauge should now read about 400 kPa.
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![](media/CO2_tanks_50.jpg)
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## 2. Turn the CO$_2$ regulator in the room
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The CO$_2$ is supplied to the incubator from the tap in the wall behind the Nikon Ti.
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* Open blue tap fully
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* Open the regulator. The gauge should read 20-40.
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![](media/CO2_wall_10.png)
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## 3. Check the water level in the humidifier bottle
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There is a bottle inside the microscope cage on the right hand side below the stage that humidifies the air coming from incubator. Check that this half full and top up if necessary.
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## 3. Turn on the Incubator
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* Turn on the incubator on from the blue switch
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* Make sure the flow rate dial is on. You should see bubbling in the humidifier bottle.
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* Set the target temperature and CO$_2$ concentration by pressing the green/red buttons to increase/decrease the targets.
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![](media/incubator_markup_20.jpeg)
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## 5. Attach the well plate cover
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The well plate cover should be in the microscope cage attached to the humidifier bottle.
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This sits on top our well plate on the stage
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![](media/wellplate_cover_markup_20.jpeg)
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![](media/wellplate_on_stage_17.6.jpeg)
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## 5. Wait for equilibrium
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It will take some time for the conditions in sample to reach their target values.
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* **CO$_2$ - 10 min**: The air above the sample will equilibrate very quickly, less than 5min.
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* **Temperature - 1 hour**: The temperature of the sample will take longer as large volume of air in the cage needs come to equilibrium. For a 15°C difference from room temperature this takes about an hour. If you need higher temperature you will need to wait longer - check current temperature in the cage on the incubator screen.
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* **Stage movements - 2 hours**: The microscope stage expands it warms up which shifts the sample slightly. Changes in Z can be corrected for using the PFS system, but the shift in XY position may affect your experiment. For high-resolution time-lapse imaging allow 2 hours for equilibration.
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![](temp_disp.png)
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# Shut down for live cell
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1. Turn off the incubator.
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2. Place well plate cover next to the humidifier bottle.
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3. Top up the humidifier bottle.
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3. Turn the regulator and tap off at the wall.
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4. Turn the tap on the CO$_2$ tank off |
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\ No newline at end of file |