ActiveMax® Human CD19 μBeads, premium grade (for cells)

DMF

Assay of human CD19 protein on the μBeads surface by Flow cytomtry. The human CD19 conjugated on the μBeads (Cat. No. MBS-C002) surface were fluorescently stained using PE labeled anti-human CD19 antibody and analyzed by flow cytometry (QC tested).

Add 100ul of 1:40 PE anti-human CD19 Antibody dilution (2.5ul stock solution in 100ul FACS buffer) into 5e5 of ActiveMax® human CD19 μBeads, premium grade (for cells) (Cat. No. MBS-C002), Negative control Beads (ActiveMax® Streptavidin μBeads, premium grade (for cells) (Cat. No. MBS-C009)) as well. PE signals was used to evaluate CD19 beads binding activity.

Add 100ul of 1:40 PE anti-human CD19 Antibody dilution (5ul stock solution in 100ul FACS buffer) into 5e5 of ActiveMax® human CD19 μBeads, premium grade (for cells) (Cat. No. MBS-C002), Negative control (ActiveMax® Streptavidin μBeads, premium grade (for cells) (Cat. No. MBS-C009)) as well. PE signals was used to evaluate CD19 beads binding activity.

ActiveMax® Human CD19 μBeads, premium grade (for cells) (Cat. No. MBS-C002) can activate CD19-specific CAR-T cells by detecting the secretion of IFN- γ in vitro (Routinely tested).

CD19 CAR-T cells were stimulated with ActiveMax® Human CD19 μBeads (MBS-C002) for 24 h , and cell-free supernatants were harvested for evaluating IFN-γ secretion by ELISA. The results showed that CD19 CAR-T cells released significantly larger amounts of IFN-γ into the supernatants in response to CD19 μBeads . Seed the CD19 μBeads in 96-well plates at a density of 2×10⁴ μBeads/well, and co-culture with CD19 CAR-T cells at an effector-to-target (E:T) ratio of 5:1, 1:1, 1:5 in 96-well tissue culture plate, respectively. CD19 CAR-T cells alone were cultured as the control.

CD19 CAR-T cells were stimulated with ActiveMax® Human CD19 μBeads (MBS-C002) for 24 h , and cell-free supernatants were harvested for evaluating IFN-γ secretion by flow cytomtry. The results showed that CD19 CAR-T cells released significantly larger amounts of IFN-γ into the supernatants in response to CD19 μBeads. Seed the CD19 μBeads in 96-well plates at a density of 2×10⁴ μBeads/well, and co-culture with CD19 CAR-T cells at an effector-to-target (E:T) ratio of 5:1, 1:1, 1:5 in 96-well tissue culture plate, respectively. CD19 CAR-T cells alone were cultured as the control.

CD19 CAR-T cells were stimulated with ActiveMax® Human CD19 μBeads (MBS-C002) for 24h, and cell-free supernatants were harvested for evaluating Granzyme B, Perforin secretion by ELISA. The results showed that CD19 CAR-T cells released significantly larger amounts of Granzyme B, Perforin into the supernatants in response to CD19 μBeads. Seed the CD19 μBeads in 96-well plates at a density of 2×10⁴ μBeads/well, and co-culture with CD19 CAR-T cells at an effector-to-target (E:T) ratio of 5:1, 1:1, 1:5 in 96-well tissue culture plate, respectively. CD19 CAR-T cells alone were cultured as the control.

CD19 CAR-T cells were stimulated with ActiveMax® Human CD19 μBeads (MBS-C002) for 24h, and cells were harvested for evaluating CD69,CD137 expression by flow cytometry. The results showed that the proportion of CD69 and CD137 in CD19 CAR-T cells was significantly increased after CD19 μBeads stimulation. Seed the CD19 μBeads in 96-well plates at a density of 2×10⁴ μBeads/well, and co-culture with CD19 CAR-T cells at an effector-to-target (E:T) ratio of 5:1, 1:1, 1:5 in 96-well tissue culture plate, respectively. CD19 CAR-T cells alone were cultured as the control.

CD19 CAR-T cells were stimulated with ActiveMax® Human CD19 μBeads (MBS-C002) for 24h, and cells were harvested for evaluating Exhaustion Marker LAG-3, PD-1 expression by flow cytometry. The results showed that the proportion of LAG-3 and PD-1 in CD19 CAR-T cells was significantly increased after CD19 μBeads stimulation. Seed the CD19 μBeads in 96-well plates at a density of 2×10⁴ μBeads/well, and co-culture with CD19 CAR-T cells at an effector-to-target (E:T) ratio of 5:1, 1:1, 1:5 in 96-well tissue culture plate, respectively. CD19 CAR-T cells alone were cultured as the control.

CD19 CAR-T cells with low gene expression were used as a starting point. The CAR positive cells were rare (~ 0.1%). After positive selection by the CD19 μBeads at a 2:4 (Beads:cells) ratio, the CAR positivity rate increased from an initial ~0.12% to 19%, corresponding to an approximately 150-fold enhancement.

Flow plots showing CD3+ CAR+ cells under magnetic enrichment with CD19 μBeads.