Animals on the 5th day after SAH were sacrificed for peripheral blood collection from the heart to prepare the single cell suspensions. Briefly, 200 μL blood was obtained from a single animal in an anticoagulant tube coated with heparin and was lysed in the three-time volume of red blood cell lysis buffer (BD) for 10 min. After centrifuging with 300 g for 5 min, the supernatant was discarded and the cellular layer was resuspended with PBS. Cell density was controlled at 1 × 106/mL. Fluorophore-labeled antibodies(including CD4 and CD25) were used following the manufacturer's instructions (ebioscience) [14 (link)]. BD FACS Calibur flow cytometer was applied.
Fluorophore labeled antibodies
Manufactured by Thermo Fisher Scientific
Sourced in United States
Fluorophore-labeled antibodies are laboratory reagents that consist of antibodies conjugated with fluorescent dye molecules. These antibodies are used in various analytical techniques, such as immunofluorescence microscopy and flow cytometry, to detect and visualize specific target molecules or proteins within biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Lsrii instrument, by BD (2 mentions)
Anti cd16 32 antibody 2.4g2 clone, by BioXCell (2 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Red blood cell lysis buffer, by BD (1 mentions)
Alexa fluor 488 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Foxp3 fixation permeabilization buffer kit, by Thermo Fisher Scientific (1 mentions)
Influx cell sorter, by BD (1 mentions)
Cd8α 53 6, by Thermo Fisher Scientific (1 mentions)
Tcrβ h57 597, by Thermo Fisher Scientific (1 mentions)
Cd4 rm4 5, by Thermo Fisher Scientific (1 mentions)
4 protocols using fluorophore labeled antibodies
1
Isolation and Analysis of Peripheral Blood Cells
2
Multiparameter Flow Cytometry Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Anti-CD16/32 (2.4G2 hybridoma supernatant) was added to block Fc receptors prior to staining, which used FACS buffer (1% BSA, 0.01% NaN3, PBS) containing fluorophore-labeled antibodies purchased from eBioScience or BioLegend. Staining antibodies included anti- CD3 (clone 145 2C11), CD4 (clone RM-4-5), CD8 (clone 53–6), CD11b (M1/70), CD11c (N418), CD27 (clone LG.7F9), CD45.1 (clone A20), IFNγ (clone XMG1.2), Ly6C (HK1.4), Ly6G (1A8), NK1.1 (clone PK136), and NKp46 (clone 29A1.4). After surface staining, cells were fixed in 2–4% paraformaldehyde for direct analysis with or without saponin treatment for intracellular staining. To amplify IL-10-gfp signal [53 (link)], fixed and permeabilized cells were stained with a rabbit monoclonal anti-GFP followed by goat anti-rabbit IgG Alexa Fluor 488 (both from Life Technologies). At least 100,000 data events per sample were collected using an LSRII (BD Biosciences). FlowJo software (Treestar) was used for analysis of flow data.
3
Flow Cytometric Analysis of Immune Cell Populations
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For flow cytometric staining, fluorophore-labeled antibodies against the following markers were obtained from eBioscience (San Diego, CA, USA) unless otherwise indicated: CD4 (RM4-5), TCRβ (H57-597), CD8 (53-6.7), PD-1 (J43), CD5 (53-7.3), IL-7Rα/CD127 (A7R34), IFN-γ (XMG1.2), Bcl2 (10C4). Antibodies were used at manufacturer’s recommended concentrations. Flow cytometric staining always used an Fc block cocktail to block nonspecific staining. Fc block cocktail consisted of 3 mL each of normal mouse, rat, and hamster serum, with addition of 0.3 mg of anti-CD16/32 antibody (clone 2.4g2, BioXCell). For intracellular staining, cells were fixed and permeabilized using the eBioscience FoxP3 Fixation/permeabilization buffer kit according to supplied protocols. Standard flow cytometric analysis was performed using a BD LSR II instrument. Flow cytometric data analysis was performed using FlowJo (Treestar software, Portland, OR, USA).
Anti-IL-7Rα treatment in vivo was carried out by biweekly intraperitoneal injection of 0.5 mg of anti-IL-7Rα clone A7R34 (BioXcell for polyclonal PD-1−/− thymocyte disease model experiments; or generated by us for mixed Marilyn experiments) or Rat IgG2a isotype control (2A3, BioXcell or generated by us) beginning on the day of cell transfer.
Anti-IL-7Rα treatment in vivo was carried out by biweekly intraperitoneal injection of 0.5 mg of anti-IL-7Rα clone A7R34 (BioXcell for polyclonal PD-1−/− thymocyte disease model experiments; or generated by us for mixed Marilyn experiments) or Rat IgG2a isotype control (2A3, BioXcell or generated by us) beginning on the day of cell transfer.
4
Flow Cytometric Analysis and Sorting
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For flow cytometric staining and sorting, fluorophore-labeled antibodies against the following markers were obtained from eBioscience (San Diego, CA, USA) unless otherwise indicated: CD4 (RM4-5), TCRβ (H57-597), CD8α (53-6.7), PD-1 (J43), Ki-67 (SolA15), CD44 (IM7), CD19 (1D3), Granzyme B (NGZB), CD45.1 (A20), CD45.2 (104), Vβ6 (RR4-7). Antibodies were used at manufacturer’s recommended concentrations. Flow cytometric staining always used an Fc block cocktail to block nonspecific staining. Fc block cocktail consisted of 3 mL each of normal mouse, rat, and hamster serum, with addition of 0.3 mg of anti-CD16/32 antibody (clone 2.4g2, BioXCell). Fixation and permeabilization were performed using the eBioscience FoxP3 Fixation/Permeabilization buffer kit (Thermo Fisher) according to the manufacturer’s protocols. For cell sorting, a BD Influx cell sorter was used controlled with Spigot software (Beckton Dickinson, Franklin Lakes, NJ, USA). Briefly, for sorting source cells were stained and resuspended in HBSS + 20% FBS + 10 mM HEPES and sorted directly into FBS supplemented with 10 mM HEPES. Standard flow cytometric analysis was performed using a BD LSR II instrument. Flow cytometric data analysis was performed using FlowJo (Treestar software, Portland, OR, USA).
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!