Plan apo λ oil objective

Briefly, whole blood from Rhesus macaque provided by Biomere (Biomere Biomedical Research Models, Inc., Worcester MA) was analyzed by flow cytometry to determine the number of DEspR+CD11b+ neutrophils. White blood cells (WBCs) were then obtained, washed and resuspended in Hank’s Balanced Salt Solution (HBSS) + 2% Fetal Bovine Serum (FBS). WBCs were counted, divided into aliquots and incubated with 10 µg/ml Alexa Fluor 568-conjugated hu6g8 antibody or Alexa Fluor 568-conjugated IgG4 isotype antibody for 20 min at 4 °C. Cells were washed to remove unbound antibody, then concentrated at to approximately 10⁸ cells/ml in RPMI, then loaded into imaging device. Live cell imaging was performed using a microfluidic chip with three parallel conjoined microfluidic channels, and a confocal microscope (Ti2-E microscope equipped with Nikon A1R HD25 point scanner and 60X Plan Apo λ Oil objective) housed within a temperature and CO₂-controlled incubator. Images were then acquired every minute for the first 9 h, and then every 5 min for 15 h thereafter, for a total of 24 h observation time. At 15 min into imaging, Sytox Green (Thermo-Fisher) was added into the imaging media for each chip at a final concentration of 1:6000. (See Supplementary Methods for detail).

See Supplementary Methods for details.
Briefly, whole blood from Rhesus macaque NHP provided by Biomere (Biomere Biomedical Research Models, Inc., Worcester MA) was analyzed by flow cytometry to determine the number of DEspR + CD11b + activated neutrophils. White blood cells (WBCs) were then obtained, washed and resuspended in Hank’s Balanced Salt Solution (HBSS) + 2% Fetal Bovine Serum (FBS). WBCs were counted, divided into aliquots and incubated with 10 μg/ml Alexa Fluor 568-conjugated hu6g8 antibody or Alexa Fluor 568-conjugated IgG4 isotype antibody for 20 minutes at 4°C. Cells were washed to remove unbound antibody, then concentrated at to approximately 10⁸ cells/mL, then loaded into imaging device. Live cell imaging was performed using a microfluidic chip with three parallel conjoined microfluidic channels, and a confocal microscope (Ti2-E microscope equipped with Nikon A1R HD25 point scanner and 60X Plan Apo λ Oil objective) housed within a temperature and CO₂-controlled incubator. Images were then acquired every minute for the first 9 hours, and then every 5 minutes for 15 hours thereafter, for a total of 24 hours observation time. At 15 minutes into imaging, Sytox Green (Thermo-Fisher) was added into the imaging media for each chip at a final concentration of 1:6000.

Appropriate culture or spore suspension dilutions were placed on agarose pads containing the appropriate medium and supplemented with 1.5% LSL-LE 8200 agarose (Lonza, Basel, Switzerland) on a microscopy slide and covered with a cover glass attached to a 125 µL Gene Frame (Thermo Fisher Scientific, Waltham, MA, USA). TLFM was performed on a Ti-Eclipse inverted microscope (Nikon, Champigny-sur-Marne, France) equipped with a 60× Plan Apo λ oil objective, a TI-CT-E motorized condenser and a Nikon DS-Qi2 camera. GFP was imaged using a quad-edge dichroic (395/470/550/640 nm) and FITC single emission filters. A SpectraX LED illuminator (Lumencor, Beaverton, OR, USA) was used as a light source, using the 470/24 excitation filter. Temperature was controlled at 30 °C (for sporulation) or 37 °C (for germination) with an Okolab cage incubator (Okolab, Ottaviano, Italy). Images were acquired using NIS-Elements software (version 4.51, Nikon) and the resulting pictures were further handled with the open source software ImageJ (version 1.54d) [24 (link)].