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 ± 3.4% and 87.3 ± 4.5%, respectively (gated on CD45; mean ± 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 µM β-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-γ (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 ± 5% of the cells expressed the mature DC marker CD83 and only 1 ± 1% of cells still expressed the monocyte marker CD14 (mean ± 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 ± 25% (mean ± 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 ± 81 and 5 ± 2 pg/mL, respectively (mean ± 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 10⁶ 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 ± 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 10⁶ 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 ± standard deviation values from leukopak fractions of n = 3 unique donors.