Efferocytosis capacity was determined as the number of cell internalized or membrane bound apMPRO neutrophils per primary macrophage at 60 minutes. Our protocol was adapted from Evans et al.20 (link) In brief, apMPRO cells were added to primary macrophages grown in glass bottom 12 well plates (Cellvis, Burlington, Canada; p12.1.5H-N) at 1 × 106 apMPRO cells per well. The apMPRO cells were either centrifuged at 300 g for 1 minute, or allowed to settle at room temperature for 10 minutes, in order to bring the apMPRO cells into contact with the macrophages. After 1 hour at 37°C, primary macrophages were washed 3× with phosphate-buffered saline (PBS). Macrophage nuclei were stained with Nuclear Green DCS1 (17550; AAT Bioquest, Sunnyvale, CA), while macrophage cell membrane was stained with wheat germ agglutinin Texas red (Invitrogen; W21405). Cells were fixed using 4% paraformaldehyde in PBS for 15 minutes. Cells were imaged on a Leica DMi8 (Leica, Wetzlar, Germany) inverted fluorescent microscope as further described herein.
Glass bottom 12 well plates
Manufactured by Cellvis
Sourced in Canada, United States
Glass bottom 12 well plates are laboratory equipment used for cell culture and analysis. They provide a transparent glass surface at the base of each well, allowing for improved optical imaging and microscopy.
Automatically generated - may contain errors
2 protocols using glass bottom 12 well plates
1
Quantifying Macrophage Efferocytosis of Apoptotic Neutrophils
2
Time-lapse Imaging of Bacterial Growth
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gfp- or mCherry-labeled isolates were grown in LB media overnight, diluted 1:100, and left to grow up to approximately OD600 = 0.4 in 3 mL LB. The bacteria cultures were diluted again to a final concentration of 1:100,000 in 1 mL of 50% LB or in cucumber or wheat root extract media in glass-bottom 12-well plates (Cellvis, United States). Image acquisition was performed using an Eclipse Ti-E inverted microscope (Nikon, Japan) equipped with a Plan Apo 40x/0.95 NA air objective. An LED light source (SOLA SE II, United States) was used for fluorescence excitation. GFP fluorescence was excited with a 470/40 filter, and emission was collected with a T495lpxr dichroic mirror and a 525/50 filter. mCherry fluorescence was excited with a 560/40 filter, and emission was collected with a T585lpxr dichroic mirror and a 630/75 filter (all filters from Chroma Technology Corp., Brattleboro, VT, United States). Images were acquired with an SCMOS camera (ZYLA 4.2 PLUS, Andor, United Kingdom) at 1-h intervals for a period of 17 h. NIS software (version 5.02, Nikon Instruments, Inc.) was used for acquisition and basic image processing.
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