Table 2.

Calculation of total volume of antibody needed for staining

	1 sample	11 samples (1 batch)	48 samples (4 batches)	Final volume to stain (+ ∼20%)
Number of cells / sample	1.5 × 106 cells	16.5 × 106 cells	66 × 106 cells
Staining volume	30 μL	330 μL	1320 μL	1600 μL

The number of cells and staining volume for one single sample is shown and multiplied by 11 samples included in one staining batch. Next, a factor 4 is used to prepare antibody cocktail for 4 batches (48 samples). To account for pipetting error and avoid touching the bottom of the tube ∼20% is added to the final staining volume. This calculation allows to easily scale up experiments to a multibatch (4 in this case) setting, preserving cell concentration used for antibody titration. It shows the staining volume needed to stain one batch but also allows to calculate total staining volume required to prepare antibody cocktails aliquots.