For phenotypic analysis of T cells, the PPD and peptides stimulated lungs and spleen/LNs cells were analysed by flow cytometry. Lymphocytes culture were harvested and stained with fluorochrome tagged anti-CD4-PE, CD8-APCCy7, CD62L-FITC, CD44-PerCPCy5.5, CD11c-PECy7, F4/80-APC, CD86-PE, CD80-FITC, CD40-PECy5, and MHC-II-PerCPCy5.5abs (BD Biosciences, San Jose, CA). Briefly, lymphocytes were harvested in tubes and washed with FACS buffer (PBS + 2%FCS). Cells were Fc blocked using anti-mouse CD16/CD32 Ab. Later, stained with fluorochrome-labelled Abs. After staining, cells were fixed by using 1% paraformaldehyde in FACS buffer. Cells were acquired in BD-FACS Aria III and BD-FACS Accuri (BD, Franklin Lakes, NJ). The analysis was performed using BD-FACS DIVA, BD-C6, and Flowjo software (BD, Franklin Lakes, NJ).
Facs accuri
Manufactured by BD
Sourced in United States, Italy
The FACS Accuri is a flow cytometry instrument designed for high-performance cell analysis and sorting. It features a compact, benchtop design and provides reliable, consistent data acquisition.
Automatically generated - may contain errors
Lab products found in correlation
Facscalibur, by BD (3 mentions)
Facsdiva, by BD (2 mentions)
Golgistop, by BD (2 mentions)
Cytofix cytoperm, by BD (2 mentions)
Fcs express version 3, by De Novo Software (2 mentions)
Pe anti vb13.1 im2021u, by Beckman Coulter (2 mentions)
Cellquest pro 4, by BD (2 mentions)
Cytofix cytoperm kit, by BD (2 mentions)
Accuri c6, by BD (2 mentions)
Annexin 5 fitc, by BD (2 mentions)
Facsaria 3, by BD (1 mentions)
Cd80 fitc, by BD (1 mentions)
Cd86 pe, by BD (1 mentions)
Anti cd4 pe, by BD (1 mentions)
Cd8 apc cy7, by BD (1 mentions)
F4 80 apc, by BD (1 mentions)
Cd11c pe cy7, by BD (1 mentions)
Cd62l fitc, by BD (1 mentions)
Cd40 pecy5, by BD (1 mentions)
Cd44 percp cy5, by BD (1 mentions)
37 protocols using facs accuri
1
Phenotypic Analysis of T Cells
2
Multiparametric Flow Cytometry Analysis
3
Isolation and Characterization of Primary Human Hepatocytes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Primary human hepatocytes were isolated from three organ donors whose livers were not used for liver transplantation, acquired from the Karolinska University Hospital, using a protocol previously described (25 (link)). The cells were then stained fresh with antibodies against MICA, MICB, HLA-ABC, CD155, ULBP-1, ULBP-2, and ULBP-3 conjugated with PE and analyzed on a BD FACS Accuri.
4
Quantitative Apoptosis Assay in A549 and HeLa Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We used flow cytometry
analysis on A549 and HeLa cells for the quantitative apoptosis assay.
As previously stated, cells were cultivated at 1.0 × 105 cells/ml and plated in 24-well culture plates (Corning) for 24 h.
Cells were cultured for further 24 h after being treated with 4 μm
of target drugs3a and 3h . For analysis,
the medium was discarded and cells were washed briefly in PBS, and
then, the trypsinization protocol was carried out. Furthermore, cells
were suspended in PBS and stained with annexin in V-FITC/PI according
to the supplier technique (Sigma, USA, catalogue number: APOAF-20TST).
After adding the reagents, the cells were allowed to incubate for
30 min at room temperature before being analyzed in PBS. The analysis
was conducted on a BD FACS Accuri (BD Biosciences, USA), and the results
were analyzed using FlowJo software (BD Biosciences, USA).42 (link)
analysis on A549 and HeLa cells for the quantitative apoptosis assay.
As previously stated, cells were cultivated at 1.0 × 105 cells/ml and plated in 24-well culture plates (Corning) for 24 h.
Cells were cultured for further 24 h after being treated with 4 μm
of target drugs
the medium was discarded and cells were washed briefly in PBS, and
then, the trypsinization protocol was carried out. Furthermore, cells
were suspended in PBS and stained with annexin in V-FITC/PI according
to the supplier technique (Sigma, USA, catalogue number: APOAF-20TST).
After adding the reagents, the cells were allowed to incubate for
30 min at room temperature before being analyzed in PBS. The analysis
was conducted on a BD FACS Accuri (BD Biosciences, USA), and the results
were analyzed using FlowJo software (BD Biosciences, USA).42 (link)
5
Flow Cytometry Analysis of Cell Signaling
6
Binding Assay of Labeled Lactoferrin
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
M.tb H37Rv and M.tb H37Rv ΔmbtB::hyg strains were cultured to log phase, and 1 × 108 cells were used per assay. Cells were washed with phosphate-buffered saline (PBS) and blocked for 1 h at 4°C with 2% BSA in SFM (serum free media). Cells were then incubated with Lf-A647 (10 μg per 100 μl of SFM) alone or in the presence of 200-fold excess unlabeled human Lf at 4°C for 2 h. Finally, cells were washed extensively with SFM and fluorescence data of 104 cells per assay was acquired using a BD FACS Accuri instrument. Experiments were repeated thrice in duplicates. Statistical significance was analyzed by Student's t-test.
7
Cell Proliferation Tracking with EdU
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proliferation rates of the established cell lines were determined by using the EdU Click-it® kit (Life Technologies, Darmstadt, Germany) according to the manufacturer’s instructions. Cell lines cultured as monolayers in 2D were analyzed when achieving 50% confluence, while cell lines cultured in 3D Matrigel® droplets were analyzed 3 days after splitting. Briefly, cells were incubated for 2 h at 37°C in pre-warmed CL medium (Supplemental Tables 3 and 5 ) supplemented with 1 mM EdU before harvesting as single cells and washing in PBS− containing 1% Bovine Serum Albumin (BSA; Sigma-Aldrich, St. Louis, MO). Afterwards, cells were fixed for 15 min at room temperature (RT) in the dark with 100 µL Click-it® fixative, washed in 1% BSA/PBS− solution and permeabilized in 100 µL of a 1× saponin-based buffer (15 min, RT, protected from light). After incubation for 30 min at RT with 500 µL of the EdU Click-it® Plus reaction cocktail containing Alexa Fluor® 647 as fluorescent dye, the samples were washed in 1× saponin-based buffer and analyzed on the BD FACS Accuri.
8
Flow Cytometric Analysis of Breast Cancer Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
3D-O matrices were enzymatically digested with collagenase (20 mg/ml for 2–3 h at 37°C) on day 4. BCa cells were isolated and identified by gating cells with a high DiO signal (excitation, 488 nm; emission, 530/30 nm). Antibodies used to evaluate hypoxic status and surface marker expression were AlexaFluor 647 conjugated anti-HIF-1 α (359706, BioLegend, CA), APC-Cy7 conjugated anti-CD8 (344714, BioLegend, CA), PE-conjugated anti-PD-L1 (393608, BioLegend, CA), PE-conjugated anti-MUC-1 (355608, BioLegend, CA), and PerCP-Cy5 conjugated anti-CD73 (344014, BioLegend, CA). Cell viability was evaluated by using a Sytox Blue live-dead fluorescent dye (S34857, Invitrogen, CA) possessing excitation, 358 nm; emission, 461 nm or Live/Dead Blue cell stain (L34962, Thermo Fischer Scientific, MA). For all analyses, a minimum of 5,000 events were acquired using BD FACS Fortessa and FACSDiva v6.1.2 software or BD FACS Accuri and BS Accuri C6 software (BD Biosciences), respectively. The BCa cell counts were always normalized to a predetermined number of counting beads (424902, BioLegend, CA), and mean fluorescence intensity (MFI) ratios for each of the targets as mentioned above studied were assessed with respect to the corresponding isotype in the BCa-DiO+ cells. The data was analyzed using FlowJo program v10 (Ashland, OR).
9
Macrophage Phagocytosis of E. coli Bioparticles
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Control or ARL11-depleted RAW264.7 macrophages (3 × 105 cells/well) were cultured in a 12-well plate tissue culture dish in DMEM supplemented with 10% heat-inactivated FBS at 37 °C with 5% CO2 in a humidified cell culture chamber. Following 24 h of LPS (1 μg/ml) treatment, culture dishes were incubated on ice for 10 min, and Alexa Fluor 488-conjugated E. coli (K-12 strain) BioParticles (E13231; Life Technologies) were added to each well at a multiplicity of infection of 10:1. To synchronize the update, the dishes were centrifuged at 300 × g for 5 min at 4 °C and further incubated on ice for 30 min to allow adherence of bioparticles to the cells. The dishes were then transferred to a cell culture chamber and incubated at 37 °C for 30 min to allow phagocytosis. After the incubation period, the dishes were washed three times in ice-cold PBS to remove excess bacteria before fluorescence measurement by flow cytometry (BD FACS Accuri; BD Biosciences).
10
Quantifying Peritoneal Neutrophils in Mice
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Six hours after LPS injection, PF was harvested by lavage of the mouse abdominal cavity with 2 ml cold PBS. The collected PF was centrifuged at 6000 × g for 5 min at 4 °C. The supernatants were stored at −80 °C before cytokine levels were measured, and the pellets were resuspended. The peritoneal cells were incubated with FITC-conjugated anti-mouse Ly-6G and PE-conjugated anti-mouse CD11b (eBioscience, San Diego, CA) in FACS buffer (PBS containing 2% FBS) for 20 min. After being washed three times, the peritoneal cells were suspended in FACS buffer. Total neutrophils in PF were detected by flow cytometry (FACS Accuri; BD Biosciences, Franklin Lakes, NJ) and were gated on Ly-6G and CD11b to ascertain the percentage of neutrophils in PF.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!