Methods in Anopheles Research - MR4

Chapter 5 : Insecticide Resistance Monitoring5.2 Microplate Enzyme Activity AssaysPage 4 of 4Sodium Acetate Buffer [NaOAc]1. Place 900 ml purified water in a glass beaker on a stirrer.2. Add 83 ml 3M sodium acetate.3. Adjust to pH 5 with glacial acetic acid.4. Adjust to 1000 ml final volume.5. Store at room temperature.Protocol for mosquito preparation:1. Kill the mosquitoes by placing them in a freezer for at least 10 minutes. Mosquitoes revive afterbrief exposures to freezing temperatures and may escape. 12. Homogenize 1 adult or pupa in 100 µl of KPO 4 buffer in a grinding tube.3. Dilute to 1000 µl final concentration with KPO 4 .4. OPTIONAL: To increase the number of assays that can be performed from each mosquito, aliquot500 µl of the homogenate into separate tubes and dilute each to 1000 ml.Loading homogenates into microplates1. At room temperature (or on ice if desired), load 100 µl aliquots of homogenate into the microplatewells in triplicate on the same plate for each enzyme assay. Use a new pipette tip for each sample.Load the first mosquito sample three wells across (A 1-3) and the next mosquito in the wells directlybelow the first (B 1-3). Continue down the plate until you reach the bottom, then shift right to the nextthree vacant set of columns and continue at the top working downward. Wells A 4-6 should containyour 9th mosquito if you have followed this pattern.2. Load the positive and negative controls into the last 6 wells on the plate.3. See specific assays for detection of various activities in the following sections. These should beperformed immediately.Notes on using multi-channel pipettors• Tips must be firmly attached by strong quick pressure of the pipettor.• Use your gloved fingers to individually check the tightness of the tips before attempting to load thepipettor with reagent.• Particular care must be taken to directly observe that each tip has loaded with the same volume asothers.• Tight-fitting tips and slow deliberate loading are essential to obtaining accurate and precise data.References:http://www.cdc.gov/ncidod/wbt/resistance/assay/microplate/index.htm1 See alternatives in chapter on Mosquito Anesthesia. Killing mosquitoes with insecticide may drasticallyaffect resistance enzymes levels measured in the assays and is not recommended.