Nile Red Staining_28017347

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title: "TheNessLab : Nile Red Staining"

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TheNessLab : Nile Red Staining

Created by Wen Chen, last modified on Sep 09, 2020

Use this protocol to stain Chlamydomonas reinhardtii cells with Nile Red to quantify neutral lipids.

Materials:

• C. reinhardtii liquid culture
• 25% (v/v) DMSO in ddH2O
• 50 ug/mL Nile Red solution: 
  • To prepare 50 uL stocks (red line on cap, 200 uL PCR tubes): add 42.15 uL 25% v/v DMSO to 50 uL stock tube
  • To prepare 500 uL stocks (labelled "500," 500 uL tubes): add 421.5 uL 25% v/v DMSO to 500 uL stock tube
• Incubator (with shaking and temperature control functions)

**Before starting any procedure below, preheat the incubator to 40°C. **

Pre-staining Procedure:

This procedure performs 2 TAP-N washes and concentrate the cell culture to a suitable concentration for staining.

1. Transfer  0.5 mL of culture to 1.5 mL conical tube. 
2. Spin tube at 2000 rcf for 5 min at 25°C using the small microcentrifuge (Eppendorf Centrifuge 5427 R).
3. Dispose the supernatant.
4. Add 0.5 mL TAP-N to the pellet.
5. Vortex until the pellet disappears and you have a homogeneous green mixture.
6. Repeat Steps 2 and 3.
7. Add 100 uL TAP-N to the pellet. 
8. Repeat Step 5.
9. Use the final product for Nile red staining.

Staining Procedure:

For each sample, you will need 1 stained sample and 1 unstained control.

1. To prepare a stained sample, add the following (in order) on a 48 well plate:
   1. 3 uL 50 ug/mL Nile Red solution
   2. 5 uL concentrated cell sample
   3. 292 uL 25% v/v DMSO
2. To prepare an unstained control, add the following (in order) on a 48 well plate:
   1. 5 uL concentrated cell sample
   2. 295 uL 25% v/v DMSO
3. Wrap the 48 well plate in foil completely. 
4. Incubate the plate in dark  at 40°C and 100 rpm for 10 min. 
5. Transfer 200 uL from the 48 well plate to a 96 well plate.
6. Wrap the 96 well plate in foil completely and keep the 48 well plate with you (just in case).
7. The sample is ready for the FACS.

High Volume Staining Procedure:

Pre-staining:

1. Preheat 25% DMSO and 500 mL glass jars to 40°C (takes up to 90 min in the incubator, so use the water bath instead). 
2. Start up Cytoflex.
3. Perform two TAP/TAP-N wash on samples such that Chlamy is suspended in fresh media.
   1. Centrifuge culture at 2000g for 5 min and decant.
   2. Re-suspend in an appropriate amount of media based on the amount of cells. (Ideally the cell density at the end of this step should exceed 36,000 cells/uL)
      1. If the lag phase culture has less than 180,000,000 cells (singlets) in total the night before, re-suspend that culture in 2 mL TAP/TAP-N. 
   3. Vortex until homogeneous.
4. Add 10 uL culture from step 2c to 240 uL TAP in 96 well plate.
5. Use Cytoflex to perform cell count of the concentrated cell sample.

Staining:

For each sample, you will need 1 stained sample and 1 unstained control.

1. To prepare a stained sample, add the following (in order) in a 500 mL glass jar:
   1. 1.2 mL 50 ug/mL Nile Red solution
   2. 2 mL concentrated cell sample:
      1.       X mL concentrated cell sample from Step 2c such that you have up to 72,000,000 cells in the whole staining mix+ (2 mL - X mL) TAP/TAP-N
   3. 116.8 mL 25% v/v DMSO
2. To prepare an unstained control, add the following (in order) 500 mL glass jar:
   1. 2 mL concentrated cell sample (see equation above)
   2. 118 mL 25% v/v DMSO
3. Wrap the glass jars in foil completely. 
4. Incubate the plate in dark  at 40°C and 100 rpm for 60 min. 

Prepare for FACS analysis:

1. Add 50 mL staining mix to a 50 mL Falcon tube.
2. Centrifuge at 2000g for 5 min and decant.
3. Repeat 1 and 2. 
4. Add the remaining ~20 mL staining mix to the same Falcon tube. Repeat 2.
5. Re-suspend pellet in 1000 uL filtered 1:1 TAP:cell sorting buffer.
6. Pipette to mix until homogeneous and transfer to 15 mL Falcon tube. The sample is ready for the FACS analysis.

1. Autoclave the 1.5 mL conical tube if you still need to grow the same cell culture.
2. The pre-staining procedure can be performed on the lab bench only if you will not need the cell culture again.
3. The stained cultures will remain stable for at least a few hours after staining as long as they stay in the dark.

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