24 well cell imaging plate

Immunofluorescence microscopy was carried out by seeding out BV-2 microglia in a 24-well cell imaging plate (Eppendorf) at a density of 5 × 10⁴ cells/ml. Thereafter, cells were stimulated with S1 (100 ng/ml) for 24 h. After stimulation, cells were washed with PBS and fixed with 500 µl formaldehyde (4%) for 15 min at room temperature. This was followed by PBS washing and blocking with 500 µl of 5% bovine serum albumin for 60 min at room temperature. Cells were then incubated overnight at 4 °C with either Iba-1 Alexa Fluor® 488 antibody (Santa Cruz Biotechnology; 1:500) or Toll-like receptor 4 (TLR4) antibody (Santa Cruz Biotechnology; 1:500). In experiments employing unconjugated primary antibody, cells were washed with PBS and further incubated in the dark for 2 h with Alexa Fluor® 488–conjugated donkey anti-rabbit IgG secondary antibody (Thermo Scientific; 1:500). Then, cells were stained with 50 nM of 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI; Invitrogen) for 3 min and washed. Images were captured using EVOS FLoid® cell imaging station (Invitrogen).

Following treatment, microtissues were rinsed in PBS, fixed in formalin for 15 minutes at room temperature, rinsed in PBS twice, and then stored in PBS at 40C until ready to image. Before imaging, microtissues were switched to ScaleS4 containing 1:1000 Hoechst 33342 and 1:200 rhodamine-phalloidin. ScaleS4 is composed of 40 w/v% D-(−)-sorbitol, 10 w/v% glycerol, 4M Urea, 0.2 w/v% Triton X-100, and 15 v/v% DMSO in deionized water. After 3 hours, ScaleS4 was removed. Agarose hydrogels were removed from a 12-well plate, placed on a paper towel, the extra agarose was removed from the sides, and then flipped over into a 24-well cell imaging plate (Eppendorf) containing 50 uL of fresh ScaleS4. Cell imaging was performed using an Opera Phenix^™ High Content Screening System (Perkin Elmer) using a 20x water objective (NA 1.0, HH1400421, PerkinElmer). Image stacks were taken with a 5 µm step size. A 3D image screening protocol was set up to obtain the 3D image of the MCF7 microtissues.

Following treatment, microtissues were rinsed in PBS, fixed in formalin for 15 min at room temperature, rinsed in PBS twice, and then stored in PBS at 4 °C until ready to image. Before imaging, microtissues were switched to ScaleS4 containing 1:1000 Hoechst 33,342 and 1:200 rhodamine-phalloidin. ScaleS4 is composed of 40 w/v% D-(-)-sorbitol, 10 w/v% glycerol, 4 M Urea, 0.2 w/v% Triton X-100, and 15 v/v% DMSO in deionized water. After 3 h, ScaleS4 was removed. Agarose hydrogels were removed from a 12-well plate, placed on a paper towel, the extra agarose was removed from the sides, and then flipped over into a 24-well cell imaging plate (Eppendorf) containing 50 uL of fresh ScaleS4. Cell imaging was performed using an Opera Phenix™ High Content Screening System (Perkin Elmer) using a 20 × water objective (NA 1.0, HH1400421, PerkinElmer). Image stacks were taken with a 5 µm step size. A 3D image screening protocol was set up to obtain the 3D image of the MCF7 microtissues.