For the confirmation of engraftment, 100μl of blood was collected from both Rag1 and BLT mice via tail vein bleed and treated with heparin. Blood was blocked with 7μl mouse blocking buffer [Normal mouse serum (Jackson ImmunoResearch Labs, West Grove, PA)), Rat anti-mouse CD16/CD32 (Mouse FC Receptor Monoclonal), Human gamma globulin (Jackson ImmunoResearch Labs)] for 5–8 minutes. 3μl each of the following monoclonal antibodies: FITC-conjugated anti CD45, PE-conjugated anti-CD3, and PE-Cy5-conjugated CD4 were added to each sample, vortexed, and incubated for 30 minutes at RT in the dark. Samples were lysed and washed following the Human Erythrocyte lysing kit and cells were fixed in 1% paraformaldehyde/DPBS for flow analysis. Samples were acquired using BD Acurri C6 flow cytometer (BD Biosciences, San Jose, CA) and analyzed using FlowJo V.X. software (Tree Star, Ashland, OR).
Acurri c6 flow cytometer
Manufactured by BD
Sourced in United Kingdom, United States, France
The Acurri C6 is a flow cytometer designed for quantitative analysis of cells and particles. It utilizes laser-based technology to detect and analyze the physical and fluorescent characteristics of individual cells or particles suspended in a fluid stream.
Automatically generated - may contain errors
Lab products found in correlation
Normal mouse serum, by Jackson ImmunoResearch (1 mentions)
Anti cd8 percp cy5 5 53 6, by Thermo Fisher Scientific (1 mentions)
Accutase, by Thermo Fisher Scientific (1 mentions)
Ab225, by Abcam (1 mentions)
Mca1557a488t, by Bio-Rad (1 mentions)
C11 bodipy, by Thermo Fisher Scientific (1 mentions)
Mitosox, by Thermo Fisher Scientific (1 mentions)
Dihydroethidium dhe, by Beyotime (1 mentions)
2 7 dichlorodihydrofluorescein diacetate dcfh da, by Beyotime (1 mentions)
X vivo 20 medium, by Thermo Fisher Scientific (1 mentions)
Interleukin 7 (il 7), by Immunotools (1 mentions)
Interleukin 2 (il 2), by Immunotools (1 mentions)
Modfit lt program, by Verity Software House (1 mentions)
Annexin 5 kit, by BD (1 mentions)
Anti fas pe, by BD (1 mentions)
Anti mhc 1 fitc, by BD (1 mentions)
Anti mica b pe, by R&D Systems (1 mentions)
12 protocols using acurri c6 flow cytometer
1
Engraftment Confirmation via Flow Cytometry
2
NOD.β2m^null.HHD Mice Immune Cells
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Single-cell suspensions were made from spleens and pancreas-infiltrating immune cells of 12-week-old non-diabetic female NOD.β2mnull.HHD mice immunized with different peptides and resuspended in RPMI 1640 medium. Fluorochrome-conjugated antibodies specific for surface markers used in this study were anti-CD3-FITC (145-2C11), anti-CD4-PE (GK1.5), and anti-CD8-PerCP-Cy5.5 (53-6.7) (eBiosciences, San Diego, CA, USA). Events were collected on the BD Acurri C6 flow cytometer and analyzed with FlowJo software.
3
Erythrocyte Lysis by Plasmodium
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Plasmodium were treated with a range of SLO units as described above. Flow cytometry was used to assess equal volumes of each sample for every experiment on a BD Acurri C6 flow-cytometer (BD Biosciences, San Jose, CA.). Total erythrocyte values were obtained by gating for intact erythrocytes based on forward and side scatter. Infected erythrocyte values were obtaining by gating for SYBR-Green positive intact erythrocytes. Uninfected erythrocyte values were obtained by subtracting infected erythrocyte values from total erythrocyte values. Percent lysis of uninfected and infected erythrocyte populations were determined by comparing the infected and uninfected erythrocyte values for each SLO-treated sample in an experiment to the untreated control.
4
Characterization of Mesenchymal Stem Cells
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TSPCs at passage 1–2 were detached using Accutase (Life Technologies Ltd., Paisley, UK) and counted. Aliquots containing 1 × 106 cells were blocked with 10% normal serum in FACS buffer (2.5% FBS in PBS) for 20 min before washing. Cells were resuspended in either fluorescently conjugated or unconjugated primary antibodies for 45 min at 4°C. Anti‐CD90 (ab225, Abcam, Cambridge, UK), anti‐CD105 (MCA1557A488T, Serotec, Oxford, UK), and anti‐CD73 (550256, BD Biosciences, Oxford, UK) were used in this study. Cells were washed and either analyzed directly (CD105) by flow cytometry (BD Acurri C6 flow cytometer, BD Biosciences, Oxford, UK) or incubated with a secondary antibody for a further 45 min at 4°C (CD90 and CD73) before washing and analysis. Cells in the absence of antibody and in the presence of the secondary antibody only were used as controls. A threshold gating out at least 95.5% of the control cells was used and for samples including primary antibody the percentage of positive cells calculated as that exceeding the threshold.
5
Multimodal ROS Detection Assay
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Total ROS was detected with 2, 7-dichlorodihydrofluorescein diacetate (DCFH-DA, Beyotime Biotechnology, Nanjing, China; 10 μΜ) for 20 min; lipid ROS was detected with C11-Bodipy (ThermoFisher Scientific, Waltham, MA, USA; 2 μΜ) for 30 min; mitochondrial ROS was detected with MitoSox (ThermoFisher Scientific, Waltham, MA, USA; 10 μΜ) for 15 min, and superoxide free radicals were detected with dihydroethidium (DHE, Beyotime Biotechnology, Nanjing, China; 5 μM) for 10 min at 37 °C in an incubator. The BD Acurri C6 flow cytometer (BD Bioscience, San Jose, CA, USA) was used to detect the fluorescence intensity (MFI); ROS level was expressed as the ratio fold to the control.
6
Evaluating Anti-Plasmodium Falciparum Drug Potency
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P. falciparum was propagated in
RPMI-1640 containing 0.5% albumax I as previously described.20 (link),22 (link) For EC50 determination, parasites (0.19 mL of 0.5% parasitemia,
0.5% HCT) were plated into 96-well microtiter plates containing 10
μL compound or DMSO control. The last column of each plate was
reserved for non-parasitized RBCs (0.5% HCT) to determine background
fluorescence. Serial dilutions of compound stocks were prepared in
100% DMSO at 200× the final concentration. After 72 h of incubation,
parasitized RBCs were quantitated by the SYBR Green method. 2×
SYBR Green I solution (20 μL) in 1× PBS was mixed with
20 μL of parasites in 96-well plates and incubated for 20 min,
after which time 160 μL of 1× PBS was added. Fluorescence
was detected using a BD Biosciences Acurri C6 flow cytometer, and
events were recorded within gates that encompassed all asexual growth
stages of the P. falciparum intraerythrocytic
life cycle. A minimum 50 000 total events were recorded per
well. Background events determined from non-parasitized RBC controls
were subtracted from final counts. All data were collected in triplicate.
RPMI-1640 containing 0.5% albumax I as previously described.20 (link),22 (link) For EC50 determination, parasites (0.19 mL of 0.5% parasitemia,
0.5% HCT) were plated into 96-well microtiter plates containing 10
μL compound or DMSO control. The last column of each plate was
reserved for non-parasitized RBCs (0.5% HCT) to determine background
fluorescence. Serial dilutions of compound stocks were prepared in
100% DMSO at 200× the final concentration. After 72 h of incubation,
parasitized RBCs were quantitated by the SYBR Green method. 2×
SYBR Green I solution (20 μL) in 1× PBS was mixed with
20 μL of parasites in 96-well plates and incubated for 20 min,
after which time 160 μL of 1× PBS was added. Fluorescence
was detected using a BD Biosciences Acurri C6 flow cytometer, and
events were recorded within gates that encompassed all asexual growth
stages of the P. falciparum intraerythrocytic
life cycle. A minimum 50 000 total events were recorded per
well. Background events determined from non-parasitized RBC controls
were subtracted from final counts. All data were collected in triplicate.
7
Allogeneic Mixed Lymphocyte Reaction with moDCs
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Allogeneic MLR was performed as described previously [22 (link)] by adding 2 × 105 monocyte-depleted allogeneic PBMCs labeled with CFSE, using VybrantTM CFDA SE Cell Tracer Kit (Cat. no. V12883, Invitrogen), to 5 × 104 treated moDCs for 5 days in X-Vivo 20 medium (Cat. no. 04-448Q; Lonza) supplemented with 50 U/mL of IL-2 (Cat. no. 11340023; Immunotools) and 10 ng/mL of IL-7 (Cat. no. 11340073; Immunotools). CD3/CD28-beads (Cat.no. 11161D, Gibco, Waltham, MA, USA) were used as positive control and CFSE-stained lymphocytes only as negative control. The cells were harvested on day 5 and analyzed on an Acurri C6 flow cytometer (BD Biosciences).
8
Cell Cycle Analysis by Flow Cytometry
9
Apoptosis Evaluation of A. montana Extract
10
TUNEL Assay for Cell Apoptosis
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