Phycoerythrin pe conjugated annexin 5

Samples from healthy donors were processed first depleting CD3⁺ cells using the RosetteSep® Human Monocyte Enrichment Cocktail kit (StemCell Technologies, Vancouver, Canada) prior to a ficoll-hypaque (Rafer, Zaragoza, Spain) density gradient separation. Subsequently, CD14⁺ cells were isolated by positive selection using the EasySep® Human CD14 Positive Selection Kit (StemCell) following manufacturer's instructions. For the isolation of monocytes from MS patients, peripheral blood monocytes were isolated from 50 mL of whole blood by ficoll-hypaque density gradient separation, followed by the abovementioned CD14 positive selection step. The initial CD3⁺ cells depletion step was not performed due to the limited amount of blood. Samples were acquired on a FACSCanto II flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA), and cell viability was determined using 7-amino-actinomycin D (7-AAD) (BD Biosciences) and phycoerythrin (PE)-conjugated annexin V (Immunotools, Friesoythe, Germany) staining for 20 min at 4°C, protected from light. Cell counts were quantified using PerfectCount microspheres (Cytognos, Salamanca, Spain) and monocyte purity was determined using forward and side scatter gating strategies on FACSDiva software (BD Biosciences).

To obtain monocytes, buffy coat samples from the Banc de Sang i Teixits (Barcelona, Spain) were obtained following the institutional standard operating procedures for blood donation.
First, samples were depleted of CD3⁺ cells using RosetteSep^® Human Monocyte Enrichment Cocktail kit (StemCell Technologies, Vancouver, Burnaby, BC, Canada) followed by a ficoll-hypaque (Rafer, Zaragoza, Spain) density gradient separation. After that, isolation of CD14⁺ cells was performed by using EasySep^® Human CD14 Positive Selection kit (StemCell) following the manufacturer’s instructions. Finally, viability and cell counting of viable cells were determined by staining (20 min at 4 °C in dark) with 7-amino-actinomycin D (7-AAD) (BD Biosciences, Franklin Lakes, NJ, USA) and phycoerythrin (PE)-conjugated annexin V (Immunotools, Friesoythe, Germany) and the use of PerfectCount microspheres (Cytognos, Salamanca, Spain) for cell quantification. Samples were acquired on a FACSCanto II flow cytometer and analyzed using the FACSDiva software (BD Biosciences).

Healthy donor buffy coat samples were processed first depleting CD3⁺ cells using the RoseetteSep® Human Monocyte Enrichment Kit (StemCell Technologies, Vancouver, Canada) prior to a density gradient separation using ficoll-hypaque (Rafer, Zaragoza, Spain). Afterwards, CD14⁺ cells were isolated using the EasySep® Human CD14 Positive Selection Kit (StemCell), according to the manufacturer’s instructions. Cell viability was determined using 7-amino-actinomycin D (7-AAD) (BD Biosciences, Franklin Lakes, NK, USA) and phycoerythrin (PE)-conjugated annexin V (Immunotools, Friesoythe, Germany) staining for 20 min at 4°C, protected from light, and cell counts were quantified simultaneously using PerfectCount beads (Cytognos, Salamanca, Spain). Samples were acquired on a FACSCanto II flow cytometer (BD Biosciences), and monocyte purity was determined using forward and side scatter gating strategies on FACSDiva software (BD Biosciences).