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Allow the kit reagents to come to room temperature for 30 minutes. Ensure that all samples have reached room temperature and have been diluted as appropriate prior to running them in the kit. Standard Preparation Label glass test tubes as #1 through #7. Pipet 450 μL of water into tube #1 and 250 μL into tubes #2-#7. Add 50 μL of the Formaldehyde stock solution to tube #1 and vortex completely. Take 250 μL of the formaldehyde solution in tube #1 and add it to tube #2 and vortex completely. Add 250 μL of tube #2 to tube #3 and vortex completely. Repeat this serial dilution for tubes #4 through #7. The concentration of formaldehyde in tubes 1 through 7 will be 200, 100, 50, 25, 12.5, 6.25 and 3.125 μM. Water will be used as a sample blank. Use all Standards within 2 hours of preparation.
Urine samples containing visible protein or particulates should be centrifuged or filtered prior to using. Urine samples must be diluted 1:4 with water by taking one part of sample and adding 3 parts of water prior to using in the kit. Any urine samples with formaldehyde concentrations outside the standard curve range should be diluted further with water to obtain readings within the standard curve. TCM samples should be diluted in TCM and read off a standard curve generated in the same TCM. Use all diluted samples within 2 hours of preparation.
We recommend that all standards and samples be run in duplicate to allow the end user to accurately determine formaldehyde concentrations. 1. A plate layout sheet has been included on the back page of the insert to aid proper sample and standard identification. Set plate parameters for a 96-well Corning Costar 3694 plate. 2. Pipet 50 μL of samples, water as the blank or standards into wells in the black plate. 3. Add 25 μL of the DetectX® Formaldehyde Reagent to each well using a repeater pipet. 4. Gently tap the sides of the plate to ensure adequate mixing of the reagents. Cover the plate with the plate sealer and press to seal adequately. 5. Incubate at 37 °C for 30 minutes. Room temperature incubation will yield approximately 75 % of the fluorescent signal generated with 37 °C incubation. 6. Read the fluorescent signal from each well in a plate reader capable of reading the fluorescent signal at 510 nm with excitation at 450 nm. Please contact your plate reader manufacturer for suitable filter sets. 7. Use the plate reader's built-in 4PLC software capabilities to calculate formaldehyde concentrations for each sample.
Average the duplicate FLU readings for each standard and sample. Create a standard curve by reducing the data using the 4PLC fitting routine on the plate reader, after subtracting the mean FLUs for the zero standard. The sample concentrations obtained should be multiplied by the dilution factor to obtain neat sample values.