Cyan flow cytometer

Bead-free CD3⁺ cells were purified from CRC cell suspension by positive immunomagnetic selection (Dynabeads® FlowComp™ Human CD3, Life Technologies Ltd, UK). Purity was >95%. Cells were washed and suspended in RPMI + 0.1% bovine serum albumin (BSA) and incubated overnight with either 1 μM Maraviroc or an equal volume of DMSO vehicle, prior to the transwell assay. RPMI + 0.1% BSA either alone or containing 20 ng/ml recombinant CCL4 (Peprotech Inc, Rocky Hill, New Jersey, USA) was placed in the bottom of a 96-well transwell plate (Corning Inc, Corning, New York, USA). CD3⁺ cell suspension was placed in the upper compartment of the transwell and the plate was incubated at 37°C 5% CO₂ for 4 hours. Migrated and non-migrated cells were harvested, labelled with antibodies against CD4, CD25 and CD127 and analysed using the CyAn flow cytometer after addition of 20 μl AccuCheck counting beads (Life Technologies Ltd, UK). The chemotactic index for the absolute number or percentage of a lymphocyte subset was reported:
$$\mathit{\text{Chemotactic index}}=\frac{\mathit{\text{The absolute number (or percentage) of a lymphocyte subset that migrated in response to chemokine}}}{\mathit{\text{The absolute number (or percentage) of the subset that migrated to media alone}}}$$

Whole blood was collected from heathy nude mice. RBCs were separated by centrifugation (1000 G, 5 min) and washed twice with cold phosphate-buffered saline (PBS, pH = 7.4). Then 100 μL of packed RBCs were resuspended in 900 μL PBS and mixed with 3 μL of 0.1 M biotin-X-NHS at 4 °C for 20 min. Subsequently, the cells were washed three times with PBS (1000 G, 5 min) to remove unbounded biotin molecules. The resulting biotinylated RBCs were mixed with 200 μL of 1 mg mL⁻¹ neutravidin (Thermo Scientific) at 4 °C for 1 h. After another wash cycle with PBS (1000 G, 5 min, 3 times), the RBCs-biotin-neutravidin were mixed with the biotinylated P-FRTs for 1 h to yield P-FRT-RBCs. To quantify the molar ratio of P-FRTs to RBCs, IRDye800-labeled P-FRTs were used for the preparation of P-FRT-RBCs; the absorbance at 780 nm (maximum absorbance for IRDye800) was measured and the reading was subtracted from the absorbance of parent RBCs. The integrity of RBC cell membranes in P-FRT-RBCs was assessed by Annexin V binding assay based on a protocol provided by the vendor (FITC conjugate, Life Technologies) with a Beckman Coulter CyAn flow cytometer. The ¹O₂ quantum yield of P-FRT-RBCs was measured with a singlet oxygen sensor green reagent (S36002, Invitrogen) as a ¹O₂ indicator, by comparing to that of P-FRTs.