Precellys 24 for homogenization of mouse dorsal root ganglia

Experimental Techniques
1

Does anyone use the Precellys 24 for homogenization of tissues to make protein lysates from mouse DRG or other neural tissues?

We have it. I’ve been using it and comparing to mortar-pestle (as @tberta taught me way back when), and the Precellys underperforms the mortar-pestle.

Some recent results:

Sample inputs are L3,L4,L5 DRG from a single animal, so not a ton of tissue, but a typical experimental situaiton for us.

Sample Conc (mg/ml) Method Buffer
61_DRG_Rt 0.77 Grinder CHAPS
62_DRG_Rt 0.55 Grinder CHAPS
63_DRG_Rt 0.77 Grinder CHAPS
61_DRG_Lt 0.12 Precellys CHAPS
62_DRG_Lt 0.15 Precellys CHAPS
63_DRG_Lt 0.12 Precellys CHAPS
64 DRG_Rt 0.84 Grinder RIPA
65_DRG_Rt 0.40 Grinder RIPA
64 DRG_Lt 0.25 Precellys RIPA
65_DRG_Lt 0.23 Precellys RIPA

You can see the Precellys, no matter the buffer, is several fold less.

We’ve tried different parameters.

For the above, we used CK-14 tubes, 0.5 ml volume, with the following:

200 ul of lysis buffer.

Program:
RPM: 6500
Cycles: 2
Cycle duration: 20s
Delay between cycles: 30s

Anyone have thoughts?

@vanja I saw your recent paper using the Precellys. Are you pleased with its performance?

@sshiers @thicunha @AubinMoutal @runDRG @ShanTan @Jmwirigi

2

I never used this. To make protein lysates from tissues, I do old school sonication. 100% intensity, 5s pulses all on ice. It works for everything. For when i need to isolate synaptosomes or to do protein-protein interactions, i use a dounce homogenizer. good luck!

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3

We use the Precellys for RNA and protein extractions and it seems to work well in our hands. For human or mouse DRG tissues, I use the Precellys Soft Tissue Homogenizing beads but for other tissue types such as the sciatic I usually opt for the metallic beads. As for the buffers, I typically use RIPA, or the Tissue Protein Extraction Reagent spiked with protease and phosphatase inhibitor cocktails.

Typical Precellys procedures is as follows:

RPM: Highest speed setting.

We have the older version of the Precellys homogenizer with just low, medium, or high settings.

Cycles: 4

You can increase the number of cycles as needed.

Cycle Duration: 30s – 45s

Delay between cycles: 30s

And like @AubinMoutal, we also use old-school sonication, and it works pretty well too! Hope this helps.

Good luck,

Juliet

4

Thanks @Jmwirigi! Very helpful
What kinds of volumes of buffer are you using?
Are you putting on ice in between cycles?

5

Depending on the tissue quantities, my volumes can range from 100-450ul. Since it looks like you are homogenizing only 3 mouse DRGs (been there!), I would also suggest decreasing the lysis volume to maybe 100ul or 150ul. Note: 100uL lysis buffer may be a little tricky to homogenize. To help with this, I use the Precellys 200ul tubes with a screw cap and I find it really helps.

And yes, I put them on ice in between cycles. Our homogenizer is also kept in the cold room.

6

Thanks! Yup I’m using between 100-200 ul for that quantity of mDRG.
I use the CK14 tubes in the 0.5 mL volume.

I will keep messing around with volumes and cycles until we get it right. I want this to work. I like the throughput and the ease of just using beads, but we need to improve extraction so that’s comparable with hand grinding.

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7

I hope you are sorted with the protocol but would still like to add on few things from the protocol that I have been using.

  1. I take L3, L4, L5 DRGs from both left and right side, so in total 6 DRGs from a mice.
  2. From the moment I start to dissect out the DRGs until I get a protein lysate everything is kept on ice.
  3. For 6 DRGs, I use 100ul of RIPA buffer (always add fresh protease inhibitor to it).
  4. Homogenization: I use dounce glass tissue homogenizer (20 strokes) followed by bath sonication (5sec X 3). Let it sit on ice for 15 mins. Centrifuge at high speed for 5-10 mins at 4C. Collect the sup.

Hope this helps!!

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8

We have a Precellys evolution in the Donnelly Lab. I was wondering if you ever arrived at a homogenization protocol for mouse DRG which yielded good results? Thanks!