▲ CRITICAL STEP Ensure that the cells are triturated gently and dissociated to single cells. Large clumps will reduce transfection efficiency. 21Take a 10-μl aliquot from the cell suspension and dilute it into 90 μl of D10 medium for counting. Count the cells and calculate the number of cells and the volume of suspension needed for transfection. We typically transfect 2 × 105 cells per condition with the Amaxa SF cell line 4D-Nucleofector X kit S, and we recommend calculating for 20% more cells than required to adjust for volume loss in subsequent pipetting steps. Transfer the volume needed (20 μl per transfection plus waste volume) into a new Falcon tube.22Spin down the cells from Step 21 at 200g for 5 min at room temperature.23Prepare the transfection solution by mixing the SF solution and S1 supplement supplied in the Amaxa SF cell line 4D-Nucleofector X kit S; a total of 20 μl of supplemented SF solution is used per transfection. Likewise, we recommend calculating for 20% more volume than required.24Remove the medium completely from the pelleted cells from Step 22,
