• HOME
  • Products
  • Bone Resorption Assay Plate/Kit Frequently Asked Questions

Bone Resorption Assay Plate/Kit Frequently Asked Questions


print
Q:    After incubation of cells (5-6 Days), what is the optimum detection time on the fluorescence. When will the fluorescence start decreasing? Is there a limit to when the fluorescence must be measured? 
A: The optimum detection time is changeable depending on the cell line and culturing condition. So, we recommend you to take a small amount of conditioned medium sequentially, and to investigate the rising curve of fluorescence intensity.
  
Q:  What is the expiration date of the plate?
A:  1 year from the Shipping Date.
  
Q: Will the fluorescence of/on the plate decrease over time? 
A:  The fluorescence intensity of FACS which binds to CaP plate is stable without light.
   However, a part of FACS will elute into the medium under the existence of medium or PBS buffer. 
  
Q:  If I want to trypsinise the cells for qPCR, will the Ca-P affect the trypsinization or affect the qPCR results? 
A:  Unfortunately, we do not have any experience, and do not have any data available.
  
Q: How do I measure the pit? Is it similar to the TRAP assay? Does it measure the depth and width of the pit? (as measuring area of the pits will not be equal to the volume of the pit consumed by the oesteoclasts) 
A: Pit assay (bone resorbing activity) is not similar to the TRAP assay.
   In the pit assay, the area of the cells is measured by using the photo of the plate after removing the cells as mentioned in our manual. As you said, accurately, the volume should be measured in the pit assay. However, the measurement of volume is difficult to perform. Thus, pit area or pit number is measured usually.
  
Q:  How do I wash the cells? With Buffer? For the pit assay after the measurement of fluorescence intensity?
A:  Please treat the plate with 5% sodium hypochlorite for 5 min to remove the cells from the plate, and then wash the plate with water and dry.
  
Q:  Can do time course measurement on the plate using live cells imaging machine such as TECAN?
A: Usually, we collect the conditioned medium to the new 96 well plate and measure the fluorescence intensity. We have never measured fluorescence intensity during the culturing.
  
Q: Can I use another medium that is phenol red free during the steps of the assay that require phenol red free medium? Would it negatively affect the cells? Do you have a substitution recommendation for what medium she use during these phenol red free requiring steps? It looks like this step, seeding the cells on the CaP coated plates, takes 5 days, and she is worried that a phenol red free DMEM or other medium would not have appropriate composition to support the cells for these five days. She assumes that she can use a phenol red containing medium during the cell propagation before seeding onto the special plates . She’ll be following the protocol very closely, and will start with RAW264.7 cells (murine macrophages), and then use U937 human cells later on.
   Additionally, she noted that the kit says “5% sodium hypchlorite” solution. She wanted to know if this is included in the kit, or if you have a recommendation where she purchase this item.
   Just to clarify, the customer was noting that she needed to use a phenol red free medium during the last stage, and that there is no phenol red free medium available. Do you have a recommendation for a phenol red free medium? Or can she use any phenol red free medium during that last stage? She was worried because the last stage is 5 days, and she didn’t want to compromise the cells.
A:  
In our culture system, there are no problems for culturing for  6 days without changing the medium.
If the customer is afraid of 6 days culturing, we recommend  her to try the same culturing with normal plastic plates and confirm whether osteoclast induction is possible.
Unfortunately, we don't have any experiences using other culturing medium except for DMEM/F-12AαMEM.  Therefore, we would like customer to try the above preliminary examination.
There is no problem for the customer to use medium containing phenol red at the stage of previous culture, and changes medium to  phenol red free medium at the time of assay.
5% sodium hypochlorite is not included in this kit.
Although we use solution of Wako Pure Chemical Industries, Ltd.(Cat.197-02206), any manufacturer's sodium hypochlorite can be used for this kit, because it is just used to resolve and remove the cells.
In our culture system, there are no problems for culturing for  6 days without changing the medium.
    If the customer is afraid of 6 days culturing, we recommend  her to try the same culturing with normal plastic plates and confirm whether osteoclast induction is possible.
    Unfortunately, we don't have any experiences using other culturing medium except for DMEM/F-12AαMEM.  Therefore, we would like customer to try the above preliminary examination.
    There is no problem for the customer to use medium containing phenol red at the stage of previous culture, and changes medium to  phenol red free medium at the time of assay.
    5% sodium hypochlorite is not included in this kit.
    Although we use solution of Wako Pure Chemical Industries, Ltd.(Cat.197-02206), any manufacturer's sodium hypochlorite can be used for this kit, because it is just used to resolve and remove the cells.
     
Q:   If there is a 6-well-plate, would you accept special orders?
A:   Yes, it is possible. Please contact us directly for details.
     
Q:   Is it possible to do normal bone marrow osteoclast differentiation assay with this plate? Can I stain with TRAP at the end of the experiment as usual? 
A:   This plate "BONE RESORPTION ASSAY PLATE" is a calcium phosphate (CaP)-coated plate used to measure the bone resorption activity of osteoclasts.The bone resorption activity of the osteoclast that differentiates from osteoclast precursor cell (bone marrow or RAW264.7 cell line etc.) can be evaluated. Please note the selection of the cell when osteoclast precursor cell from bone marrow is used. In bone marrow experiment, the experiment might not go well when there are a lot of impurities other than osteoclast precursor cell. Moreover, when inducing to the osteoclast, an adequate concentration of M-CSF and RANKL might have to be examined.
   
    For the TRAP-Staining, we do not have the experiment experience, but the experiment simultaneously ( bone resorption assay and TRAP-staining) might be possible. Because the cell adheres to the plate after steoclast does resorption. If a clear result is requested, we recommend the single experiment (general culture plate). 
     
    By the way,@we are selling the following products. These products might help your research. 
     
    Osteoclast Culture Kit - RANKL (Human), Recombinant
    http://www.cosmobio.co.jp/export_e/products/cell_tissue_culture/pmc_20120327.asp?entry_id=9030
     
    TRAP Staining Kit
    http://www.cosmobio.co.jp/export_e/products/cell_tissue_culture/pmc_20110518.asp?entry_id=7209
     
    Calcification Evaluation Set
    http://www.cosmobio.co.jp/export_e/products/kits/elisa/calcification_evaluation_set.asp?entry_id=11306
     
Q:   Is it compatible with doing transfections? Will it disrupt the coating? If it is compatible, which transfection method is recommended for use with this product (he mentioned Hyperfect, Fusion6, or oligofectamine as options)?
A:   There seems to be no transfection experiment example of cell culture in this plate(calcium phosphate (CaP)-coated).   --- developer or reference paper
     
    Coated-CaP adheres considerably strongly.So, basically, it doesn't peel off if there is no acid or no strong physical irritation.
     
     As at present, details of customer's experiment are uncertain. However, if the customer's experiment is an experiment with siRNA, we recommend the use of this CaP-plate after transfection. Because the nucleic acid adsorbs calcium phosphate(CaP) .--- The transfection efficiency worsens.
     
     If possible, please inform us of details of customer's experiment purpose etc. Because the necessity for doing transfection in this CaP-plate is not clear. ---  This plate is expensive compared with a general plate. In general, screening might be necessary to obtain a target cell after transfection. At that time, a lot of plates are necessary. 

     
Q:   Are the plates stored at RT or 4C? I know we say 4C or below, but he said the actual product itself said room temperature on the box and was confused.
A:   This plate can be kept by both the room temperature and refrigeration. In the label of a present plate, It is described in the label of this plate, "Store at room temparature or below 4C in the dark". 
    ---  Because this plate is a component of the kit, and to avoid confusing. 
     
Q:   If he is not using all the wells at once, can the plate be used again? I mentioned some concern if the whole plate was cultured at 37C. Could you confirm?
A:   Basically, we do not guarantee such use. But, unused well will be able to be used if there are no contamination etc. --- However, coated-CaP reacts with the moisture of the CO2-incubator, and there is a possibility that properties(quality) change a little. 
     
Q:   Does it work for Osteoclasts as you have tested here Raw cells? How do you measure pit assay resorption using image analyzing software? Can you give more details? 
A:   Unfortunately, we do not have any experience of tests using differentiated (matured) osteoclasts. It would be difficult if other cell are contaminated, but we recommend you to try and test, and get actual results.
     
    Please see attached  document regarding "Method of Measuring Pit Area". 
    (http://www.cosmobio.co.jp/export_e/products/uploads/document/Method_of_Measuring_Pit_Area_110615-2.pdf)
    However, settings will differ depending on the photography apparatus and photography condition.
     
Q:   I want to test it several times without using up one plate (24 well) at once. Since recommended preservation methods are below 4 degrees Celsius and are shading, I worry about the influence on unused plate after having tested it once in an incubator (37 degrees) for about 7 days. As it is in an incubator, it is a dark place, but it will be under light at the time of analysis for a short time. Please tell me how much effect these have if you know.
A:   It may be possible to use this product in several batches only when it is without contamination, and is a germfree state. However, it is expected that making an assay several times in such situation will be difficult as experimental maneuver. Unfortunately, we do not recommend using it in several batches. Also, we assume no responsibility for experimental results and the quality deterioration resulting from the use of the product in several bathces. We would highly appreciate it if you could understand about this.
     
Q:   What type of mineral is the calcium phosphate? How is the calcium phosphate coated on the plate?
A:   Unfortunately, the information is not open to public.

To be used for research only. DO NOT use for human gene therapy or clinical diagnosis.


“Bone Resorption Assay Plate/Kit Frequently Asked Questions” also belongs to the following categories.