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Inquiry: Dear Sirs, Can this product, Cl.diff. Toxin B Protein ab12401, also be used as a positive control in a Cytotoxicity Assay (measuring cytopathic effect in cell culture)? Thank you and kind regards
Asked on Jul 19 2012
Thank you for contacting us again.
I am delighted to send you the protocol of the cell toxicity assay with Vero cells
1 Verocycotoxicity Assay
1.1 Vero cells are plated out the night before on a 96-well plate at a concentration of 20K cells per well (in 100ml), then incubated at 37°C, 5% CO2. The following day they should be lightly confluent.
1.2 Lyophilised toxin/toxoid is resuspended as per the instructions in the certificate of analysis, and diluted in duplicate to 0.1mg/ml in DMEM (200ml total) without FBS in the first well of a non-TC-treated 96-well plate. A serial 5-fold dilution is performed (40ul in 160ul) in the same media using a multichannel for 11 dilutions in total, leaving the 12th dilution as a media control.
1.3 20ul of each dilution is then pipetted onto each well of the pre-prepared Vero cells and then incubated at 37°C, 5% CO2 for 24 hours.
1.4 The following day, the cells are examined using an inverted phase contrast tissue culture microscope. Cells with active toxin on should be rounded, cells without significant toxicity should look like the control cells.
Of course the protocol dilutions may be altered as desired by the customer to suit their specific application.
Answered on Jul 19 2012