Linear uptake rates were determined over 5 min. Samples were removed after 1,2,3 and 5 min, transferred to 4 ml of ice-cold buffer, filtered on glass fiber filters, and washed twice with 4 ml of buffer.
Samples for electron microscopy analyses were fixed in paraformaldehyde (final concentration 2.5%, w/v), washed 3 times for 15 min in sodium phosphate buffer (pH 7.4) and filtered on a 0.2 µm filter.
Marrow was flushed from the femora with 5 6 ml of ice-cold FACS buffer, filtered through a 40-μm cell strainer and kept on ice.
The pellet was re-homogenized in modified RIPA buffer; filtered through a 0.22 µm syringe filter and stored at −80°C.
The concentrated solution was filtered on a column (2.0 × 65 cm) of Toyopearl HW55S equilibrated with the same buffer.
Briefly, cells were resuspended in binding buffer at 1×106 cells/ml and stained with Annexin V and propridium iodide for 15 min, diluted 1∶1 with binding buffer, filtered, and counted immediately on a FACScalibur flow cytometer.
Blood samples (10 ml) were processed within 4 h, diluted 1 : 10 in an erythrocyte-lysis buffer and filtered on the ISET device.
All of the buffers were filtered on 0.2 µm pore size membranes (Fisher Scientific, ON, Canada).
Within 4 h after venopunction, blood samples were diluted 1 : 10 in an erythrocyte lysis buffer containing paraformaldehyde, filtered on the ISET device using filters with calibrated pores of 8 μ (Vona et al, 2000; Paterlini-Brechot and Benali, 2007).
For LILRA3 D1 and D1D2, the buffer was exchanged gradually to BIAcore buffer (HEPES, pH 7.4) and finally filtered on 200 10/300 GL column (GE Healthcare).
