AID EliSpot assay tutorials – PBMC isolation part 1

Cell isolation via density gradient In this tutorial, we guide you through the first steps of PBMC isolation—an essential procedure for EliSpot assays and many immunological applications. The video covers everything from preparing your materials to correctly layering diluted blood over Ficoll under sterile conditions. You’ll learn how to dilute whole blood with PBS, gently mix the sample, and overlay it onto Ficoll using either a pipette or a direct tilt-transfer method. The tutorial also explains how to calculate reagent volumes, why temperature matters, and which laboratory equipment is required for a smooth workflow. By the end, you’ll clearly see how to create the two distinct layers - Ficoll at the bottom and diluted blood on top - setting the stage for successful PBMC separation. Whether you're new to PBMC isolation or refreshing your technique, this step-by-step walkthrough ensures a solid and safe start to the process. #EliSpot #Immunology #PBMC #PBMCIsolation #DensityGradient #LabTutorial ⏱️ TIMESTAMPS 0:00 Welcome 0:06 Introduction: PBMC isolation overview 0:15 Summary of steps 0:20 Step 1: Diluting blood with PBS 0:29 Pipette vs. tilt-transfer methods 0:35 Resulting two-layer setup 0:43 Sterile conditions required 0:51 Equipment & reagents needed 1:19 Allow reagents to reach room temperature 1:28 PBMC isolation timing guidelines 1:35 Preparing Ficoll tubes 1:46 Example volume calculation 2:00 Diluting blood in a new tube 2:17 Adding PBS (volume proportions) 2:44 Mixing gently 2:50 Overlaying diluted blood on Ficoll 3:02 Pipette transfer technique 3:33 Adjusting tube position 3:53 Verifying two layers 4:06 Direct tilt-transfer technique 4:35 Completing the transfer 4:54 Checking layer integrity 5:05 Closing remarks