Colorimetric assay with TRAP staining with human Osteoclasts

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jackclj
jackclj's picture
Colorimetric assay with TRAP staining with human Osteoclasts

Hi all,
  I've been told that the differenciation of osteoclasts from human peripheral blood samples could be quantified by a colorimetric assay specific to differentiatted osteoclasts. I guess it would involve TRAP staining and an absorbance plate reader but I  can't find a protocol anywhere  to confirm that and perhaps save me work. Does anyone have ever seen an assay similar to this.
Thank you
Christian

Rajeshwari patel
Rajeshwari patel's picture
I think this will help you

I think this will help you

TRAP Staining Kit

Tartrate-resistant acid phosphatase staining of osteoclasts

Cat. No. AK04

For Research Use Only

Notice to Purchaser
This product is to be used for Research Purposes Only. It is not to be used for Drug or Diagnostic Purposes, nor is it intended for Human Use. B-Bridge products may not be resold, modified for resale, or used to manufacture commercial products without the express written consent of B-Bridge International, Inc.
EXCEPT AS OTHERWISE EXPRESSLY SET FORTH IN THIS USER MANUAL, B-BRIDGE DOES NOT MAKE ANY REPRESENTATION OR WARRANTIES OR CONDITIONS OF ANY KIND, EITHER EXPRESS OR IMPLIED, WITH RESPECT TO THE PRODUCTS, OR INFORMATION DISCLOSED HEREUNDER, INCLUDING, BUT NOT LIMITED TO, THE IMPLIED WARRANTIES OF MERCHANTABILITY, FIT FOR A PARTICULAR PURPOSE, OR NONINFRINGEMENT OF THE INTELLECTUAL PROPERTY RIGHTS OF THIRD PARTIES.

© 2006, B-Bridge International, Inc. All Rights Reserved.

TRAP staining kit

Introduction

The TRAP Staining Kit (cat. #AK04) is used for the staining of tartrate-resistant acid phosphatase in osteoclasts. Bone mass is controlled by the balance between the activity of osteoblasts and the activity of osteoclasts. Alkaline phosphatase and tartrate-resistant acid phosphatase are used as markers for osteoblasts and osteoclasts, respectively. The TRAP Staining Kit supplements the Bone Reabsorption Assay Kits.
Our TRAP Staining Kit uses the same buffer as our Acid Mucopolysaccharide Assay and our DNA Quantity Assay therefore a single same can be shared among these assays.

List of Components

Store the complete kit at 4ºC

     

    1 kit contains reagents for staining 10 x 96-well plates

     

    • ??

     
    Fixative, 10% Formalin, neutral buffer (Reagent 1) 1 bottle, 50 mL
     

    • ??

     
    Tartrate-containing Buffer, 50 mM, pH 5.0 (Reagent 2) 1 bottle, 50 mL
     

    • ??

     
    Chromogenic Substrate (Reagent 3) 10 vials, 3 mg/vial
    Additional Materials Required

       

       

      • ??

       
      Microplate reader or spectrophotometer capable of reading absorbance at 540 nm
       

      • ??

       
      Distilled or deionized water (dH2O)
       

      • ??

       
      Pipets
       

      • ??

       
      37°C incubator
      Protocol

      Staining Procedure (96-Well Plate)

         

        • 1. Remove culture medium. Wash each well once with 100 μL of PBS.

         
         

        • 2. Add 50 μL of the Fixative (Reagent 1) to each well and fix for 5 minutes at room temperature.

         
         

        • 3. Wash each well 3 times with 250 μL of dH2O.

         
         

        • 4. Dissolve 1 vial of Chromogenic Substrate (Reagent 3) with 5 mL of Tartrate-containing Buffer (Reagent 2).

         
         

        • 5. Add 50 μL of Chromogenic Substrate to each well.

         
         

        • 6. Incubate at 37°C for 20 to 60 minutes (only osteoclasts are stained as shown in figure 1).

         
         

        • 7. Wash with dH2O water when the best color condition is obtained.

         

        Note:
        Excess incubation will cause precipitation so be sure to stop reaction before precipitation starts.
        B-Bridge International, Inc. 2 V060707A www.b-bridge.com TRAP staining kit

        Quantitation of TRAP in Culture Supernatants

           

           

          • 1. Dissolve 1 vial of Chromogenic Substrate (Reagent 3) with 5 mL of Tartrate-containing Buffer (Reagent 2).

           
           

          • 2. Dispense 30 μL/well of culture supernatants into 96-well plate and add 170 μL/well of the Chromogenic Substrate/Tartrate-containing buffer prepared above.

           
           

          • 3. Incubate at 37ºC for 3 hours.

           
           

          • 4. Read in a microplate reader at 540 nm.

           

          Figure 1
          TRAP staining of osteoclasts
           
          Figure 2
          TRAP activity measured in osteoclasts culture supernatant

          B-Bridge International, Inc. 3 V060707A www.b-bridge.com TRAP staining kit

          Companion Kits

          Primary Precursor Osteoclasts Culture Kits for Rat and Mouse
          Rat Catalog # OSC21, OSC22, and OSC25
          Mouse Catalog # OSC23 and OSC24
          For questions please contact

          B-Bridge International, Inc

          320 Logue Avenue, Mountain View, CA 94043, USA
          Tel: 650-969-7727 Fax: 650-969-7737
          Email: customersupport@b-bridge.com
          www.b-bridge.com

          B-Bridge International, Inc. 4 V060707A www.b-bridge.com

          jackclj
          jackclj's picture
          It looks even more simple

          It looks even more simple than I thought it would be! I'll try that with the reagents I already have. Thank you for the info!
          Chris