The concentration of the target cells was measured using a haemocytometer by the trypan blue exclusion method. answer (which is usually where where = is the constant of proportionality which is usually itself proportional to the probability of binding. The solutions to this equation are saturation type curves which have an initial value of d(the initial binding rate when is still small) of levels off at where ~5 m is the radius of a cell, and treating the attached nanobeads conjugated to antibodies as discs of radius ~80 nm, it would appear that the maximum quantity of beads on a cell surface would be 4is the maximum portion of the cell surface area that can be covered by beads without overlap. Assuming MAIL that for 20 min at 20 C. PBMC were collected at the interface and then washed twice with PBS at 300 for 10 min at 20 C. Cell concentration was determined by haemocytometer (Boeco, Hamburg, Germany) using the trypan blue (Gibco, Life Technologies, Stockholm, Sweden) exclusion method. 4.2. Characterization of Paramagnetic Nanobeads The 150 nm HMX anti-human anti-CD3, anti-CD14, and anti-biotin magnetic beads were from X-Zell RG14620 Biotec, Bangkok, Thailand. According to the manufacturer, antibodies were conjugated to carboxyl-functionalized polysaccharide beads made up of a multi-domain magnetite core by carbodiimide chemistry. The size distribution, morphology, and crystallinity of the nanobeads were determined by dynamic light scattering (DLS), transmission electron microscopy (TEM), and X-ray diffraction (XRD), respectively. For the DLS, the bead suspension was analysed in a Zetasizer (Malvern Devices Ltd., Worcestershire, UK). For the TEM, an aqueous answer of the nanobeads was dispersed on a copper grid, dried under vacuum, and micrographs were recorded using a Hitachi-600 electron microscope at 80 kV. The XRD experiment was performed using a Rigaku (TTRAX III) X-ray diffractometer with fixed monochromater at a wavelength and velocity of 0.1542 nm and 3/min, respectively. The amount of antibody around the beads was determined by a Bradford assay. Briefly, antibody-conjugated nanobeads were placed in Bradford answer for 60 min and the protein concentration was determined using a NanoDrop RG14620 spectrophotometer ND-1000 (Thermo Fisher Scientific, Waltham, MA, USA) at 595 nm. 4.3. Isolation of Untouched CD3- and CD14-Positive Cells 4.3.1. Magnetic LabelingUntouched CD3- or CD14-positive cells were isolated from PBMC using buffer-optimized HGMS, anti-biotin magnetic beads, and a biotinylated antibody cocktail. The cocktail contained anti-CD14, -CD16, -CD19, -CD123, -CD235a for untouched CD3-positive cells, and anti-CD3, -CD7 -CD16, -CD19, -CD56 -CD123, -CD235a for untouched CD14-positive cells. All reagents were from X-Zell Biotec, Bangkok, Thailand. Briefly, PBMC were resuspended in HGMS buffer (3% BSA/PBS, pH 7.4) and incubated with human TruStain FcX (BioLegend, San Diego, CA, USA) (5 L per 2 106 cells) for 5C10 min at 4 C to block Fc receptors. 10 L of biotinylated antibody cocktail (for untouched CD3- or CD14-positive cells) was added and incubated for 10 min at 4 C. Finally, anti-biotin magnetic beads were mixed and incubated for 15 min at 4 C. The incubation combination was shaken every 5 min by finger tapping and finally washed (300 at 4 C for 10 min). The incubation volume was managed at 250 L. New, filtered, chilly buffer (3% BSA/PBS, pH 7.4) was used in the assay. 4.3.2. Magnetic Isolation of Untouched CD3- and CD14-Positive CellsMagnetically labeled PBMC were resuspended in HGMS buffer (3% BSA/PBS, pH 7.4) (500 L/107 cells) and subjected to magnetic separation as described previously [27]. Briefly, the HGMS column was filled with HGMS buffer. Air flow bubbles were removed by gentle finger tapping. The HGMS column was placed inside an HGMS magnet 5 min before loading the sample. The HGMS column was connected to a 26G/?-inch needle via a stopcock. Magnetically labelled PBMC were loaded onto the column while the stopcock was opened. The column was washed with 8C10 mL buffer (0.5% BSA/PBS, pH 7.4) at a circulation rate of 0.33 mL/min. The target cells (untouched CD3- RG14620 or CD14-positive cells) were allowed to circulation through. The flow-through was centrifuged and target cells were pelleted at 300 for 10 min at 4 C. The concentration of the target cells was measured using a haemocytometer by the trypan blue exclusion method. Fresh, filtered, chilly buffers (3% BSA/PBS or 0.5% BSA/PBS; pH 7.4) were used in the assay. The HGMS columns and magnet were from X-Zell Biotec, Bangkok, Thailand. 4.4. Circulation Cytometry The purity of the untouched CD3- or CD14-positive cells was decided using a FACScan circulation cytometer (BD, Erembodegem, Belgium). Cells were labelled with anti-CD3 or anti-CD14 antibodies (Exbio, Prague, Czech Republic) conjugated with phycoerythrin (R-PE) or fluorescein (FITC) (Innova Biosciences, Cambridge, UK). Cells were analysed before and after magnetic separation to confirm enrichment. 10,000 events were acquired from each.