Phrodo red e coli bioparticles conjugate

Phagocytosis was performed using pHrodo Red E. coli BioParticles Conjugate (ThermoFisher Scientific cat#P35361). pHrodo is a fluorogenic dye that exhibits a dramatic increase in fluorescence at an acidified pH pHrodo-labelled bacteria show minimal basal fluorescence [(Kapellos et al., 2016 (link)) see Figure 7A], but upon phagocytosis by Dictyostelium and transport to lysosomes, detected fluorescence increases (Figure 7A). For some experiments, WT Dictyostelium were first stimulated with either buffer control, 50 nM cAMP, or 50 nM folate at 6 min intervals for 1 hr to mimic progressive chemotactic relay-response. The secretion of PDE and folate de-aminase prevents the accumulation of either chemoattractant.
The phagocytosis assay was performed in a 96 well plate. ~0.9×10⁵Dictyostelium in 100 μL PB were added to a select well and allowed to adhere to the well base for 1–5 min. Subsequently, ~10 μg of pHrodo Red E. coli BioParticles in 100 μL PB was added to each well. Fluorescent [Excitation/Emission (nm) 560/585] images were acquired and processed at regular time intervals (~10 to~15 min). Data were exported, analyzed, and graphed using Microsoft Excel.

Three to seven days old adult females were injected with 69 nL of latex beads (Thermo Fisher Scientific), 16% w/v (re-suspended in PBS and sonicated prior injection). Control flies were injected with the same volume of PBS. Twenty-four hours post-injection, flies were injected with 69 nL pHrodo™ Red E. coli BioParticles™ Conjugate (Thermo Fisher Scientific, P35361). The phagocytic activity was observed under a fluorescence microscope (Zeiss Imager.M2) after 30 min. The red fluorescence was quantified in fields of same size. Ten flies of each line were scored in each experiment and three independent experiments were performed.

Phagocytosis was measured by incorporation of bioparticles, as previously reported^{31 (link)}. For this, 2 × 10⁵ cells were incubated with 1 μg of pHrodo Red E. coli BioParticles Conjugate (Thermo Fisher, Darmstadt, Germany) for 90 min at 37 °C, and phagocytosis was assessed by flow cytometry (BD Fortessa, Heidelberg, Germany).

Phagocytosis of bacterial particles was assessed using the pHrodo™ Red E. coli Bioparticles® Conjugate for phagocytosis according to the manufacturer’s protocol (Thermo Fisher Scientific). Primary microglia were pretreated with aleglitazar (125 μM) or vehicle (DMSO) for 24 h. Excitation wavelength was 550 nm and the fluorescence of the pHrodo™ Red conjugate was collected at 620 nm using a plate reader (TriStar LB941, Berthold Technologies).

Macrophage differentiation of HSCs was carried out using the published protocol with minor modifications (23 (link)). Briefly, control and edited HSCs were plated in non-tissue culture treated polystyrene plates with macrophage differentiation medium (SFEM-II, SCF (100ng/ml), Flt3-L (50ng/ml), IL-6 (10ng/ml), IL-3 (10ng/ml), GM-CSF (10ng/ml) and M-CSF (10ng/ml). Non-adherent cells were collected every 72 hours and reseeded in the macrophage differentiation medium. Adherent cells were cultured using RPMI medium containing 10% FBS along with GM-CSF (10ng/ml) and M-CSF (10ng/ml). After 14-16 days, adherent cells were observed under microscope for morphology, harvested with Accutase (Material Number: 561527, BD Biosciences), stained for CD4 PE, CD14 FITC, CCR5 APC, CXCR4 APC, CD14 BV421, CD80 FITC, CD206APC, CD64 PE, CD163-PE CF594 and CD71 FITC antibodies and analyzed using BD FACS Aria III flow cytometer. Phagocytic potential of generated macrophages was validated using pHrodo Red E. coli BioParticles conjugate (Cat No, P35361, Thermo scientific) as per the manufacturer’s instructions. The proportion of phagocytosis was calculated by enumerating the phagocytosis positive and negative cells.

biogel-elicited peritoneal macrophages were obtained from wild type and Mc3r−/− mice 4 days after an i.p. injection with 1 ml of 2% biogel (BioRad, Hemel Hempstead, UK) [27 (link)]. 1 × 10⁶ cells were plated in 24-well plates and pre-treated with corresponding drugs for 30 min before addition of bacteria (pHrodo™ Red E. coli Bioparticles^® Conjugate, Invitrogen, Paisley, UK). After 20 min, non-ingested bacteria were washed and cells incubated for further 30 min to allow fluorescence to develop. Cells were trypsinized and analyzed by flow cytometry (BD FACSCalibur™, FL-2).