S. pyogenes strains AP1, MC25, BM27.6, BMJ71 (1×107 CFU) in GVB++ were coincubated with indicated concentrations of chimeric FH proteins in the presence of 10% FCS at 37°C, 5% CO2 for one hour. Bacteria were harvested and washed with 1× PBS. Subsequently, bacteria were stained with Cell Trace Calcein Violet (Molecular Probes) and stained with goat anti-human IgG-Alexa Fluor 488 (Invitrogen; to detect FH-hIgG1 proteins) or goat anti-mouse IgG-Alexa Fluor 647 (Invitrogen; to detect FH-mIgG2a proteins) for 30 minutes. Thereafter, bacteria were washed twice with 1× PBS and resuspended in 1x PBS. Binding of fusion proteins to bacterial surface was measured using cyflow space flow cytometer (Partec) and geometric mean fluorescence intensity (gMFI) was calculated with FlowJo software (Tree Star). To assess serum FH binding, bacteria were similar treated, however incubated in 10% NHS and stained with MRC OX-24 anti-human FH conjugated to Dylight 650. To identify bacteria, cell trace violet positive events were gated and further analyzed.
Celltrace calcein violet
Manufactured by Thermo Fisher Scientific
CellTrace Calcein Violet is a fluorescent cell-permeant dye used to label and track cells. It can be used to monitor cell proliferation and viability.
Automatically generated - may contain errors
Lab products found in correlation
Propranolol, by Bio-Techne (2 mentions)
Chloroquine, by Enzo Life Sciences (2 mentions)
Isoproterenol, by Bio-Techne (2 mentions)
Cyto id autophagy detection kit 2, by Enzo Life Sciences (2 mentions)
Etomoxir, by Bio-Techne (2 mentions)
Torin 1, by Cell Signaling Technology (2 mentions)
Mouse catalog 713704 and human catalog 713604 gm csf, by BioLegend (2 mentions)
Mouse catalog 713502 and human catalog 713402 g csf, by BioLegend (2 mentions)
Mouse catalog i9646, by Merck Group (2 mentions)
Human catalog i1395 il 6, by Merck Group (2 mentions)
Propranolol catalog p0884, by Merck Group (2 mentions)
Isoproterenol catalog i6504, by Merck Group (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Alexa fluor 647 goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 goat anti human igg, by Thermo Fisher Scientific (1 mentions)
Lsm700 axio examiner z 1 confocal microscope, by Zeiss (1 mentions)
Plan apochromat x 20 1.0 dic water objective, by Zeiss (1 mentions)
Zen 2010, by Zeiss (1 mentions)
Collagenase, by STEMCELL (1 mentions)
Aqua fluorescence reactive dye, by Thermo Fisher Scientific (1 mentions)
5 protocols using celltrace calcein violet
1
Quantifying FH Binding to S. pyogenes
2
Mucus Penetrability Assay for Colon Tissue
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The mucus penetrability assay was performed on distal colon tissue as previously described [16 (link),37 (link)]. Briefly, the colonic explants were mounted in the perfusion chamber with basolateral Kreb’s glucose solution containing 1μg/ml Calcein violet tissue stain (CellTrace Calcein Violet, Molecular Probes, Thermo Fisher) and a suspension of far red fluorescent beads (FluoSpheres Carboxylate-Modified Microspheres, 1.0 μm, red fluorescent (580/605), 2% solids, Molecular Probes, Thermo Fisher) and Kreb’s mannitol solution was added to the apical side and allowed to sediment for 5 min before it was refilled with Kreb’s mannitol solution. The distribution of the beads in the mucus was investigated by acquiring confocal images in XY stacks (320 x 320 μm) 30min after tissue mounting. These were obtained using a LSM700 Axio Examiner Z.1 confocal microscope with Plan-Apochromat x 20/1.0 DIC water objective (Zeiss) and the ZEN 2010 software (Zeiss). Average bead distance from the tissue surface was calculated by combining bead fluorescence intensity data for each z-plane above the tissue surface.
3
Investigating Immune Modulation in Preclinical Models
4
Investigating Immune Modulation in Preclinical Models
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Mouse (Catalog I9646) and human (Catalog I1395) IL-6 were from MilliporeSigma. Mouse (Catalog 713704) and human (Catalog 713604) GM-CSF and mouse (Catalog 713502) and human (Catalog 713402) G-CSF were purchased from Biolegend. The T cell proliferation dye used was CellTrace Calcein Violet (ThermoFisher Scientific, Catalog C34858). propranolol (Catalog P0884) and isoproterenol (Catalog I6504) were purchased from MilliporeSigma. In vitro, propranolol was used at a concentration of 1 μM and isoproterenol was used at a concentration of 10 μM. Etomoxir (Tocris, Catalog 4539) was used in vivo at a dose of 10mg/kg and in vitro at a concentration of 10 μM. To stain MDSCs for autophagic vesicles, the CYTO-ID® Autophagy detection kit 2.0 (Catalog ENZ-51031-0050) from Enzo Life Sciences was used. LPS (MilliporeSigma, Catalog L3024) was used at a concentration of 10ng/ml. Torin-1 (Cell Signaling, Catalog 14379S) was used at a concentration of 250 nM. 3-MA (Torics, Catalog 3977) was used at a concentration of 2 mM. Chloroquine (Enzo Life Sciences, Catalog 51031) was used at a concentration of 10 μM.
5
Enrichment of Mesenchymal Stem Cells from Bone Marrow
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Unprocessed samples of IC-BM and VB-BM aspirates were seeded onto Vitoss™ (Stryker® UK Limited, Berkshire, UK) as described before [29 (link)]. Briefly, 400μl of BM sample was added into 100mm3 of Vitoss then incubated at 37°C with gentle rocking for 3 hours to enhance the cell attachment. Each BM sample was used to seed Vitoss in duplicate to examine the MSC attachment and survival using microscopy and flowcytometry respectively. The scaffolds were next rinsed with PBS to remove red blood cells then moved into culture plates in the StemMACS MSC Expansion media and cultured for 2 weeks. After culture, for microscopy, the scaffolds were examined for cell attachment using a SP2 TCS confocal laser scanning microscope (Leica, Buckinghamshire, UK). The DAPI (Thermo Fisher Scientific) and Phalloidin (Sigma-Aldrich) dyes were used to detect cell nuclei and actin, respectively. For flowcytometry-dependent characterisation, the scaffolds were digested using 0.25% collagenase (Stem Cell Technologies). and the released cells were characterised for the surface phenotype of MSCs as previously described [29 (link)]. The released cells were stained using CD45, CD90 and CD73 antibodies (BD Biosciences) as well as live/dead markers, CellTrace calcein violet and Aqua-fluorescence reactive dye (Thermo Fisher Scientific), to identify live MSCs.
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