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Figure 1. Typical EasySep? Human Monocyte Isolation Kit (Catalog #19359)

Starting with PBMCs prepared from human peripheral blood, the monocyte cell content (CD14+CD16-) of the isolated fraction obtained without (middle plots) or with (bottom plots) EasySep? Human Platelet Removal Cocktail is typically 89.7 ¡À 3.4% and 87.3 ¡À 4.5%, respectively (gated on CD45; mean ¡À SD for the purple EasySep? Magnet). In the above example, the purities of the start and final isolated fractions obtained without (middle plot) or with (bottom plots) the EasySep? Human Platelet Removal Cocktail are 20.0%, 88.2%, and 84.8%, respectively (gated on CD45) and 18.0%, 55.7%, and 71.7% (not gated on CD45).

Figure 2. Cryopreserved Monocytes Differentiate into Dendritic Cells and Secrete IL-12 (p70) and IL-23 Upon Activation

Monocytes freshly isolated from a leukopak (Catalog #70500) using EasySep? Human Monocyte Isolation Kit (Catalog #19359) or cryopreserved monocytes (Catalog #70034) were cultured for 6 days in RPMI 1640 Medium (Catalog #36750) with 10% FBS, 0.1 mM MEM Non-Essential Amino Acid Solution (100X, Catalog #07600), 2 mM L-Glutamine (Catalog #07100), 1 mM Sodium Pyruvate (Catalog #07000), and 50 ?M ¦Â-mercaptoethanol. Human Recombinant IL-4 (Catalog #78045) and Human Recombinant GM-CSF (Catalog #78015) were added on Days 1, 3, and 6 to differentiate monocytes into DCs. Cells were either left unstimulated (control) or stimulated with LPS and Human Recombinant IFN-¦Ã (Catalog #78020) (activated). Activation led to secretion of (A) IL-12 (p70) and (B) IL-23, which were not detectable in unstimulated controls as measured using the Human IL-12 (p70) ELISA Kit (Catalog #02014) and the Human IL-23 ELISA Kit (Catalog #02016),respectively. *Cytokine concentration of control in culture was lower than the limit of detection.

Figure 3. Mature DCs Generated with ImmunoCult?-ACF Dendritic Cell Medium with Supplements Show Desired Phenotype

Monocytes isolated using EasySep? Human Monocyte Isolation Kit (Catalog #19359) were cultured and differentiated into mature dendritic cells (DCs) as described in the PIS for ImmunoCult?-ACF Dendritic Cell Culture Kit (Catalog #10985). (A) The percentage of CD14 and CD83 expression in cells at Day 7 (mature DCs) was determined by flow cytometry. At Day 7, a total of 93 ¡À 5% of the cells expressed the mature DC marker CD83 and only 1 ¡À 1% of cells still expressed the monocyte marker CD14 (mean ¡À SD, n = 39). The yield of mature DCs was determined by the count of total viable cells at Day 7 relative to the count of viable monocytes used for initial culture at Day 0. At Day 7, the yield of viable mature DCs corresponded to 45 ¡À 25% (mean ¡À SD, n=39). (B) Immature DCs were cultured as described in Figure 1. At Day 5, cells were cultured with maturation supplement for 2 days (mature DCs) or without maturation supplement (immature DCs). Supernatant was collected at Day 7 and IL-12p70 levels were determined by ELISA. Concentrations of IL-12p70 in supernatant of mature and immature DCs were 361 ¡À 81 and 5 ¡À 2 pg/mL, respectively (mean ¡À SEM, n = 27).

Figure 4. Refrigeration of Leukopaks Preserves Monocyte-to-Macrophage Differentiation Efficiency for Up to 5 Days

Using EasySep? Human Monocyte Isolation Kit (Catalog #19359), monocytes were isolated from 1 leukopak fraction (Catalog #70500) of each storage condition daily for 5 days, and 1 x 10? isolated cells were cultured in ImmunoCult?-SF Macrophage Medium (Catalog #10961) supplemented with 50 ng/mL Human Recombinant M-CSF (Catalog #78057) for a further 6 days. (A) Representative flow cytometry plot from leukopaks stored 1 day at fridge temperature (FT), showing maintenance of CD14 and upregulation of CD68 expression over the 5 day differentiation to M0 macrophages. (B) While monocytes isolated from FT-stored leukopaks efficiently differentiated into M0 macrophages, those stored at room temperature (RT) for over 3 days failed to differentiate, as shown by low percentages of CD14+CD68+ cells. Moreover, monocytes harvested from Day 5 leukopaks stored at RT failed to thrive in the 6-day culture, resulting in very few viable cells recovered (not shown). All data points represent average ¡À standard deviation values from leukopak fractions of n = 3 unique donors.

Figure 5. Refrigeration of Leukopaks Preserves Monocyte-to-Dendritic Cell Differentiation Ability for Up to 5 Days

Using EasySep? Human Monocyte Isolation Kit (Catalog #19359), monocytes were isolated from 1 leukopak fraction (Catalog #70500) of each storage condition daily for 5 days, and 1 x 10? isolated cells were cultured in ImmunoCult?-ACF Dendritic Cell (DC) Medium (Catalog #10986) supplemented with ImmunoCult?-ACF Dendritic Cell Differentiation Supplement (Catalog #10988). (A) Representative flow cytometry plot from leukopaks stored 1 day at fridge temperature (FT), showing efficient downregulation of CD14 and upregulation of CD11c over 6 days of culture. (B) While monocytes isolated from FT-stored leukopaks efficiently differentiated into DCs, those stored at room temperature (RT) for over 3 days show reduced DC differentiation ability and CD14-CD11c+ cell output. Moreover, monocytes harvested from day 5 RT-stored leukopaks failed to thrive in the 5-day culture, resulting in very few viable cells recovered (not shown). All data points represent average ¡À standard deviation values from leukopak fractions of n = 3 unique donors.