Question

Why can sulphorhodamine-B assay (SRB assay) for preliminary anticancer screening not be done with HL-60 Cells?

I have observed that preliminary anticancer activity has been performed in most of the cases with SRB assay but when studies are carried out on HL-60 cells, only MTT is used. SRB is inhibitory and MTT is antiproliferative. Is there any specific characteristic of HL-60 cell lines which prohibit them from being used with SRB?

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  • Aiken Huang · Nankai University
    Maybe it is because that the HL-60 cells are growing in suspension. The SRB assay has fixation and washing protocols, which prohibit the use of this assay in suspension cells.
  • Alok Nahata · Dr. Harisingh Gour University
    Which one is better - SRB or MTT?
  • Galina Posypanova · Kurchatov Institute
    MTT is quite adequate method for HL-60 cells.
  • Prem Subramaniam · Columbia University
    Alok,

    The SRB assay is a total protein assay, while MTT is a cell metabolic assay. As a functional assay, one would prefer MTT. The SRB assays has been adequately used in HL-60 and similar leukemia cells, but requires centrifugation steps to collect the cells/protein after trichloroacetic acid precipitation. Also, if you use media with FBS, it is sure to be co-precipitated with cells. There is a protocol that modifies the SRB assays to accomodate for this and specifically against leukemia cells like HL-60. For these reasons the MTT assay is superior: 1. no removing media, 2. no washing or centrifugation - it is a one-pot assay.

    BUT, MTT CANNOT be used in melanoma cells like the popular B16F10, because melanin in the cells absorbs at around the same wavelength as the MTT product. In these cases, the SRB is the obvious choice. Of course, the interference in melanomas will vary and may be more obvious at lower absorbance values. A protocol for SRB is attached, if you are interested.
  • Prem Subramaniam · Columbia University
    Sorry !! here is the attachment
  • Godefridus Peters · VU University Medical Center
    Both MTT and SRB assays are growth inhibition assays, although one will often see percentage survival in the Y-axis, which is incorrect. In a 1990 paper the NCI compared both assays and these yielded similar results for the 60-cell line panel of the NCI (Skehan et al, JNCI 82, 1107, 1990). We subsequently also tested whether the SRB assay can really estimate cell kill as well and compared the MTT with SRB in a large panel of cells and found similar data (Keepers et al, Eur J Cancer 27, 897,1991). However, the SRB is just very inconvenient for suspension cells, because of the multiple centrifugation steps. However, for solid tumor cells lines, it is much more convenient, rapid, more sensitive, while one can stop at several time points. However, take care to include a day 0 (day of drug addittion) in order to observe potential cell kill, which can be observed when values go below the day 0 value. Check the NCI website for correct protocol and calculation. http://dtp.nci.nih.gov/branches/btb/ivclsp.html
  • You Can Try fruorometric celltiter blue assay. It is also related to cell metabolism and used for cytotoxic activity of anticancer agents.
  • Vakamullu Sreedhar · Life Technologies
    MTT is most sutable assay for hl-60 cellsvcytotoxicity assay.But you can also use SRB assay for cytotoxicity.
    Below reference may provides the info
    http://www.ncbi.nlm.nih.gov/pubmed/17035025
  • Kamil Brożewicz · Medical University of Gdansk
    HL-60 cell line has been successfully used in srb assay for many years in NCI
    http://dtp.nci.nih.gov/docs/misc/common_files/cell_list.html
    Good luck!
  • Alok Nahata · Dr. Harisingh Gour University
    What is the difference in terms of the mechanism of action of these assays, both of them are indicating the same activity, yet methods and mechanisms differ. If the protocols are different, results should also purport different mechanisms.
    I have done SRB for preliminary screening. But when I was required to do mechanistic studies, I was directed to use HL -60 and MTT was performed. A bit confused with this thing.
  • Prem Subramaniam · Columbia University
    MTT and its cousins MTS and WST (water soluble tetrazoliums) depend on cellular dehydrogenase activity, while SRB is a total protein assay - a dye used originally for determining protein concentration. Thus MTT depends on metabolic state of cells, while SRB just measures total protein in celsls I have attached several papers that will help you.
  • Prem Subramaniam · Columbia University
    #2
  • Prem Subramaniam · Columbia University
    #3
  • Prem Subramaniam · Columbia University
    #4
  • Alok Nahata · Dr. Harisingh Gour University
    @ Dr. Prem Subramaniam
    Dear Sir
    Many thanks for providing the relevant literature on the topic. It is really helpful for my work.
    Thanks
    Best Regards
  • Kellen Gasque · Sanford-Burnham Medical Research Institute
    You can find in Keepers et al., 1991 that althought sulforhodamine B protein assay (SRB) provided a better linearity with cell number and higher sensitivity, and it also stained recently lysed cells.
  • Godefridus Peters · VU University Medical Center
    I was one of the authors of this paper. We compared the SRB and MTT for a number of cell lines, in this paper and other papers. The IC50 values, the TGI and LC50 were similar, but SRB showed a better sensitivity and linearity. SRB can be performed on suspension cell lines such as HL-60 but is very inconvenient, since you have to spin down the cells after each step; since monolayer are attached cells, this is not necessary. SRB is een protein assay; MTT a metabolic assay, so when you start you need to continue and measure until you are ready.
  • Jason Fritz · United States Environmental Protection Agency
    It sounds like the answer to your original question is "no". I remember reading the SRB vs. MTT papers from NIEHS/NTP and others regarding HTS applications, but now I'm curious to see if the SRB is still more sensitive/precise when compared to the water-soluble forms like MTS. Promega claims that MTS is more sensitive/linear than MTT, but they sell MTS, and that claim could just be marketing.
  • Monalisa Brito · Universidade Federal da Paraíba
    These papers uploaded here are really very helpful!!!

    I don´t know if it´s the right place to ask, but it is a correlated question:
    In an in vivo assay, called "Hollow fiber assay", cell lines are put to grow into nanofibers implanted on the back and abdomen of mice, and after the treatment with the tested drug, cells are removed and "counted" with the MTT assay, because it´s a fast technique.

    Attested by all here in this topic, I conclude it could be another technique: but not SRB (because of the extended time period demanded). Which technique could be suitable, in this case?

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