Je 5.0 elutriation rotor

The mutant pool was size-fractioned using centrifugal elutriation with the Beckman JE-5.0 elutriation system. This technique separates cells on the basis of size. A tube of pooled mutant population was thawed on ice and used to inoculate 2 l of YPD at an OD₅₉₅ of 0.05. Mutant cells were grown for four generations at 30° under agitation to reach ∼5 × 10¹⁰ cells. Cells were then pelleted by centrifugation and resuspended in 50 ml fresh YPD. To disrupt potential cell clumps and separate weakly attached mother and daughter cells, the 50 ml pooled cells were gently sonicated twice for 30 sec. The resuspended cells were directly loaded into the elutriator chamber of the Beckman JE-5.0 elutriation rotor. A 1 ml sample of cells was retained separately as a pre-elutriated cell fraction. The flow rate of the pump was set to 8 ml/min to ensure the loading of cells. To elute small cell size mutant fractions, the pump flow rate was increased in a step-wise fashion (in 2–4 ml/min increments). For each flow rate, a volume of 250 ml was collected from the output line of the rotor.

White-cell concentrates were obtained from the peripheral blood of healthy human volunteers (Red Cross Blood Bank), and monocytes were removed within 24 h of collection by density gradient separation of the white blood cells on Lymphoprep (Axis-Shield, UK) followed by counterflow centrifugation elutriation using a Beckman Avanti J-26 XPI centrifuge equipped with a JE-5.0 elutriation rotor and a 4.0 mL elutriation chamber (Beckman Instruments Inc., USA) at 21°C, as described previously [18 (link)]. Collected fractions were examined by a Cytospin system (Shandon, USA) and Wrights’ stain (DiffQuik; Laboratory-Aids, Australia). Monocyte purity of >90% and viability of >95% by Trypan Blue exclusion were confirmed by light microscopy, and the monocytes were resuspended in serum-free RPMI and used immediately for chemotaxis studies.