A Semi-automated Approach to Preparing Antibody Cocktails for Immunophenotypic Analysis of Human Peripheral Blood

The overall goal of this semi-automated staining antibody cocktail preparation protocol is to establish a reliable immunophenotyping method for minimizing the variability of reagent dispensing.This method can help address key issues in the immunomonitoring field such as:how to control for the intra-and inter-experimental variability from reagent dispensing.The main advantage of this technique is that automation of the cocktail preparation increases the pipetting accuracy, while reducing the errors of manual cocktail preparation.Demonstrating the procedure will be Iliana Gonzalez, a technician from my laboratory.Before beginning the procedure, apply human readable labels to 2D barcode tubes.Next, transfer the entire contents of each vial of antibody into the appropriate corresponding 2D barcode tubes, taking care to avoid bubbles.When the antibodies have been transferred, use the 2D barcode reader to read the tubes.Then, create a spreadsheet containing the antibody names and the barcode reads, and save the spreadsheet as a comma separated values file.Now, launch the 2D barcode reader, and then the automated liquid handler.To prime the syringes of the automated liquid handler, under Instrument, select Home All Axes.Confirm that none of the syringes contain visible air bubbles.When the syringes are primed, open the methods for making the base cocktail, and the activation cocktail, in the automated liquid handler software.Next, place the microfuge tubes into the small tube rack microfuge tubes for the base cocktail and activation cocktail, accordant to the number of base and activation cocktails to be made.Use the instrument setup schematic as a reference for placing the tube racks on the deck.Place the buffer reservoir in the appropriate section of the deck, and use an eight channel screw cap decapper to decap the 2D barcode tubes containing the antibodies.To avoid cross-contamination, place each cap in a cap holding tray as they are removed, to keep the caps in the appropriate order.Then place the P50 LLS filter tips, the P200 LLS filter tips, the P1000 tips, and the Matrix 1mL tube rack on the deck in their appropriate positions.Click on the start icon to start the method.When the popup window appears, confirm that all of the items on the deck match the instrument setup schematic for the method.If everything is in position, click OK.The automated liquid handler will transfer buffer from reservoir to microfuge tubes.The Matrix 1mL tube rack will move on to the 2D barcode reader, and the barcodes will be read.When all of the barcodes have been read, click OK in the popup window, to begin the antibody transfer.Upon completion of the transfer, use the eight channel screw cap decapper to recap the 2D barcode tubes, and return the tubes to 4