Here is a protocol that we use in my lab using the sigma kit and it works quite nice:

1. Carefully wash the cells 2x with PBS to remove loosely adherent cells

2. Add 500µl fixation buffer and incubate for 3 minutes at room temp

3. Wash the cells 2x with PBS

4. Add 500µl TRAP solution and incubate for 5-15 minutes

(Evaluate under the microscope, to avoid over staining. Cells should be violet-brown. Not too pink)

5. Wash again in PBS

6. Add 500µl Glycerin /PBS 1:1 mixture to keep your cell morphology until you are done taking your beautiful pictures

Fixation solution:

0,625ml citrate

0,2ml 37% PFA

1,625ml Aceton

TRAP-stain solution:

First mix:

1,8ml dist water

20µl Naphthalon

80µl Acetat

40µl Tartrate

Then mix in a separate eppi tube:

20µl Sodium Nitrite

20µl Fast garnet

Vortex and incubate 2 min at RT

Add the two mixtures together

Always prepare the TRAP-stain solution fresh right before use

 Hope this could help...

Please refer to the paper "Mineral Trioxide Aggregate Inhibits Osteoclastic Bone Resorption" and "Mineral trioxide aggregate solution inhibits osteoclast differentiation

through the maintenance of osteoprotegerin expression in osteoblasts". They may be of some help.

Here is the protocol we use in the lab.

- Dissect long bones (tibiae and femurs) free of adhering tissues from 6-8 week old mice. Cut the proximal and distal ends. Isolate BMMs from bone marrow stromal cells by flashing with 10 ml αMEM slowly injecting at one end of the bone using a sterile needle.

- Centrifuge at 1,000 rpm to collect marrow cells, wash and remove blood cells by treatment with red blood cell lysis buffer.

- Plate cells in αMEM, 15%FBS, 1X PS and M-CSF 10 ng/ml (R&D) on non-treated plates to which stromal cells and lymphoid adhere poorly.

-After 3 days wash cells with PBS and harvest with 0.05 mM EDTA in PBS for 20 min. Count BMMs and plate 2.5X103 cell/cm2 in 6, 48 or 96 well plates in αMEM, 15%FBS, 1XPS, M-CSF 10 ng/ml (R&D) and Rankl 5 ng/ml (R&D).

-After 2 days change medium. OCL will start to appear at day3. Perform TRAP staining and isolate RNA and total protein at day4 and day5.

Hi Samra,

this protocol woks qute well in my former lab:

1. Sacrifice mouse: “sterilize” mouse with 70% Ethanol (pour all over), prepare skin, then exarticulate lower extremity above hip joint and put in sterile Petri dish (10cm);

2. prepare femura and tibiae sterile, strip all skin and muscles as good as possible;

3. cut off both ends of log bones

4. flush bone marrow with 5ml complete media per bone (25G needle) through a 70µm nylon mesh in a 50ml Falcon tube; afterwards, flush nylon mesh with additional 2ml of media;

5. pipet cell suspension few times to disaggregate cells

6. centrifuge at 400g/4°C/7min

7. resuspend cells in 5ml RBC lysis buffer (s. protocol)

8. incubate at RT for 2min – add 20-30ml PBS afterwards (to neutralize)

9. centrifuge at 400g and 4°C for 7min

10. resuspend cells in 5ml complete media, put on ice

11. count viable cells (typical yield for 2 femura 5-10x107 cells

12. plate cells at 1x106 cells/24 well (1ml per well) and add 30ng/ml M-CSF

13. after 24 hours, take non-adherent cells, count them and plate them at 1x106 cells/ml in complete media + 30ng/ml M-CSF + 50ng/ml RANKL and change media every 72hours

14. perform TRAP stain between day 4-6

Reagents

70% Ethanol

Sterile scissor and tweezer

Petri dishes, sterile, 10cm (Corning Costar)

Complete media: alphaMEM+Glutamax (Gibco, Cat.No. 32561-029)

+10% heat-inactivated FCS (Gibco)

+ antibiotics (Pen/Strep)

Sterile PBS w/o Ca and Mg (Gibco)

Falcon tubes 50ml

Cell strainer (70µm, Fa. BD, Cat.No. 3522350)

Sterile Needle 25G

Syringes 5ml

Recombinant murine M-CSF (R&D, Cat.No. 416-ML 50µg)

Recombinant murine RANKL (R&D, Cat.No. 462-TEC, 10µg)

RBC lysis buffer, sterile (see protocol)

Counting chamber for cell count

Thanks  Mario, Francesca and Li. Your help is highly appreciated.

I am looking forward to set-up the assay soon

good luck!

Dear Mario, do you have a TRAP staining protocol for culture plates. I have ordered TRAP kit from Sigma but the protocol described is for blood, bone marrow films etc.

Thanks

Dear Samra, you can use this kit also for cell culture tissue plates, works nicely!

Best,

Mario

O yes, I know it can be used for culture plates but I don't have the volumes per well and incubation time etc. with fixative, staining solution and counter stain

Dear Samra, I don't have a protocol for this, but if I remember right it was included in the kit. For the incubation time you will see when the cells are stained enough to identify them...hope that helped.

Thanks Mario for all the help

Samra, did you ever develop a protocol for TRAP staining in culture plates, or is it included with the Sigma kit?

Thanks,

Here is a protocol that we use in my lab using the sigma kit and it works quite nice:

1. Carefully wash the cells 2x with PBS to remove loosely adherent cells

2. Add 500µl fixation buffer and incubate for 3 minutes at room temp

3. Wash the cells 2x with PBS

4. Add 500µl TRAP solution and incubate for 5-15 minutes

(Evaluate under the microscope, to avoid over staining. Cells should be violet-brown. Not too pink)

5. Wash again in PBS

6. Add 500µl Glycerin /PBS 1:1 mixture to keep your cell morphology until you are done taking your beautiful pictures

Fixation solution:

0,625ml citrate

0,2ml 37% PFA

1,625ml Aceton

TRAP-stain solution:

First mix:

1,8ml dist water

20µl Naphthalon

80µl Acetat

40µl Tartrate

Then mix in a separate eppi tube:

20µl Sodium Nitrite

20µl Fast garnet

Vortex and incubate 2 min at RT

Add the two mixtures together

Always prepare the TRAP-stain solution fresh right before use

 Hope this could help...

Hi Samra m stuck with same problem. If u cud please provide the staining protocol in detail

 thanks in advance  

 Hi Mohit,

We used TRAP staining kit from SIGMA (as per recommended protocol) and it worked fine in our hands. Here is the link to the product http://www.sigmaaldrich.com/catalog/product/sigma/387a?lang=en®ion=DK