Plan apochromat 20 0.16 na air objective

Following seven days of co-culture, cancer cell coverslips were removed from the top of the PDMS ring and the monocyte derived cells (MDCs) at the bottom of the microdevice were fixed for 30 minutes using 4% paraformaldehyde. MDCs were rinsed with 0.1% BSA in PBS then permeabilized and blocked in buffer containing 0.3% Triton X-100 and 1% normal goat serum in PBS for 45 minutes at room temperature. Cells were then incubated with primary antibodies diluted in dilution buffer containing 1% BSA, 0.3% Triton-X, 1% normal goat serum, and 0.01% sodium azide for 2 hours at 25˚C [anti-CD68 (1:200, ab213363, Abcam, Eugene, OR), anti-CD163 (1:100, ab182422, Abcam)]. After rinsing with PBS, all cells were incubated with secondary antibody diluted in the same dilution buffer as the primary antibodies (1:500 goat anti-rabbit IgG Alexa Fluor 488) for one hour at room temperature protected from light, then counterstained with Hoescht 33258 for five minutes (1:1,000). All images were obtained using a Zeiss Axio Observer.Z1 inverted microscope with an AxioCam 506 mono camera, Plan-Apochromat 20 × 0.16-NA air objective, and Zen 2 software (Zeiss). ImageJ software (NIH) was used to quantify CD68+ and CD163+ counts per field of view, and percent CD68 and CD163 were calculated as the ratio of CD68+ or CD163+ counts to total cell counts from the Hoechst signal.

Following seven days of co-culture, cancer cell coverslips were removed from the top of the PDMS ring and the monocyte derived cells (MDCs) at the bottom of the microdevice were fixed for 30 minutes using 4% paraformaldehyde. MDCs were rinsed with 0.1% BSA in PBS then permeabilized and blocked in buffer containing 0.3% Triton X-100 and 1% normal goat serum in PBS for 45 minutes at room temperature. Cells were then incubated with primary antibodies diluted in dilution buffer containing 1% BSA, 0.3% Triton-X, 1% normal goat serum, and 0.01% sodium azide for 2 hours at 25˚C [anti-CD68 (1:200, ab213363, Abcam, Eugene, OR), anti-CD163 (1:100, ab182422, Abcam)]. After rinsing with PBS, all cells were incubated with secondary antibody diluted in the same dilution buffer as the primary antibodies (1:500 goat anti-rabbit IgG Alexa Fluor 488) for one hour at room temperature protected from light, then counterstained with Hoescht 33258 for five minutes (1:1,000). All images were obtained using a Zeiss Axio Observer.Z1 inverted microscope with an AxioCam 506 mono camera, Plan-Apochromat 20 × 0.16-NA air objective, and Zen 2 software (Zeiss). ImageJ software (NIH) was used to quantify CD68+ and CD163+ counts per field of view, and percent CD68 and CD163 were calculated as the ratio of CD68+ or CD163+ counts to total cell counts from the Hoechst signal.