Gating strategies can be found in Supplemental Information (Figures S7 and S8 ). For analysis of mixed chimerism, cells were first stained with LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit (ThermoFisher Scientific) and blocked with TruStain FcX anti-mouse (Biolegend) for 10 min on ice in Cell Stain Buffer (Biolegend). Antibodies used for staining from Biolegend were as follows: CD45.1 PerCp-Cy5.5 (A20), CD45.2 Pacific Blue (104), CD3 AF488 (17A2), CD4 PE (RM4–4), CD11b BV605 (M1/70), CD19 PE-Cy7 (6D5), CD49b APC (DX5), CD25 PE-Cy5 (PC61), CD8a PE (53–6.7), Ter-119 PE, CD11b PE (M1/70), Gr-1 PE (RB6–8C5), CD3 PE (17A2), B220 PE (RA3–6B2), CD317 PE (927), CD172a APC (P84), CD11c AF700 (N418), CD304 PE (3E12), FOXP3 AF647 (150D), Helios AF488 (22F6), B220 FITC (RA3–6B2), CD8a BV510 (53–6.7). eBioscience antibodies used were as follows: CD117 APC (2B8), Sca-1 Pe-Cy7 (D7). Staining of intracellular markers was conducted with Biolegend True-Nuclear Transcription Factor Buffer Set as per manufacturer’s instructions. For live cell sorting, propidium iodide (MilliporeSigma) was used to determine viability. Cells were analyzed and/or sorted with a BD FACSAria II. Data were analyzed using FlowJo (10.7).
Cd45.2 pacific blue
Manufactured by BioLegend
Sourced in United States
CD45.2 Pacific Blue is a fluorochrome-conjugated antibody that binds to the CD45.2 antigen, which is a protein tyrosine phosphatase expressed on the surface of hematopoietic cells. This antibody can be used to identify and characterize cell populations in flow cytometric analyses.
Automatically generated - may contain errors
Lab products found in correlation
Cell stain buffer, by BioLegend (2 mentions)
Live dead fixable near ir dead cell stain kit, by Thermo Fisher Scientific (2 mentions)
Sca 1 pe cy7, by Thermo Fisher Scientific (2 mentions)
True nuclear transcription factor buffer set, by BioLegend (2 mentions)
Cd3 af488, by BioLegend (2 mentions)
Anti mouse trustain fcx, by BioLegend (2 mentions)
Cd45 1 percp cy5, by BioLegend (2 mentions)
Facsaria 2, by BD (2 mentions)
Cd11b apc cy7, by BD (2 mentions)
Lsrii flow cytometer, by BD (2 mentions)
Ly6g pe cy7, by BioLegend (2 mentions)
Cd86 pecy5, by BioLegend (1 mentions)
Neural tissue dissociation kit, by Miltenyi Biotec (1 mentions)
Csf 1r bv605, by BioLegend (1 mentions)
Cd3 af700, by BioLegend (1 mentions)
Cd206 pe cy7, by BioLegend (1 mentions)
Cd45.1 apc, by BioLegend (1 mentions)
Cd3e pe, by BioLegend (1 mentions)
Kaluza v1, by Beckman Coulter (1 mentions)
Sca1 apc, by BioLegend (1 mentions)
9 protocols using cd45.2 pacific blue
1
Multiparameter Flow Cytometry Analysis of Hematopoietic Reconstitution
2
Isolation and characterization of immune cells from the brain
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immune cells were isolated from whole-brain homogenates as recently described (15 (link)). Briefly, brain tissue was processed using the neural tissue dissociation kit (Miltenyi Biotec) in accordance with the manufacturer’s specifications. Afterward, cells were separated using a discontinuous Percoll density gradient (30–37–70% Percoll layers), which results in the enrichment of all immune cell types and removes a lot of myelin, which is auto-fluorescent. Following density gradient centrifugation, immune cells were washed and stained for 30 min at 4 °C using the following antibodies: CD11b APC-Cy7 (BD Biosciences), CD45.2 Pacific blue, CD86 PE-Cy5, and CD206 PE-Cy7 (BioLegend). Respective isotype control antibodies or unstained samples were used to determine positive populations. Myelin debris and dead cells were excluded by FSC/SSC gating, and singlet populations were analyzed.
For flow cytometric analyses of blood samples, EDTA-blood was stained at 4 °C for 15 min with the following antibodies: B220 Pacific Blue, CD3 AF700, CSF-1R BV605 (BioLegend), and CD11b APC-Cy7 (BD Biosciences). For more details see Waltl et al. (15 (link)).
Flow cytometry was performed using an LSRII flow cytometer (BD Biosciences), and data were analyzed using FlowJo software (Tree Star).
For flow cytometric analyses of blood samples, EDTA-blood was stained at 4 °C for 15 min with the following antibodies: B220 Pacific Blue, CD3 AF700, CSF-1R BV605 (BioLegend), and CD11b APC-Cy7 (BD Biosciences). For more details see Waltl et al. (15 (link)).
Flow cytometry was performed using an LSRII flow cytometer (BD Biosciences), and data were analyzed using FlowJo software (Tree Star).
3
Murine Hematopoietic Cell Isolation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mice were bled into 150 µL of Alsever’s solution. Samples were then treated with 10 mL ACK buffer for 2 min and centrifuged at 1600 RPM for 5 min. The supernatant was removed, and pellets were washed in 5 mL sample media (SM, PBS with 2 mM EDTA and 2% FCS). Antibodies of Biolegend (San Diego, CA, USA): CD45.1 APC, CD45.2 pacific blue, Mac1 PE-Cy7, B220 APC-Cy7, CD3e PE, Lineage-PacificBlue (Including- anti Ter119, Mac1, Gr1, CD3e, CD4, CD8 and B220), c-Kit Alexa780, Sca1-APC, CD150-PEcy7, CD48- Percp cy5.5, CD9-APC, CD34-PE, CD84-PE. Cells were stained on ice for 1 hr and washed. Flow cytometry analysis of the reporter expression frequencies (ZsGreen+) and the surface markers’ expression were performed on the Gallios Flow Cytometer (Beckman Coulter, Brea, CA, USA). Fluorescence activated cell sorting (FACS) data analysis was performed using Kaluza v1.2 (Beckman Coulter). Sorting was performed on FACSAria III (BD), as previously reported [9 (link)].
4
Isolation and Analysis of Immune Cells
5
Multicolor Flow Cytometry of Murine Immune Cells
6
Multiparameter Flow Cytometric Analysis
7
Comprehensive Immune Cell Phenotyping
8
Multiparameter Flow Cytometry Analysis of Hematopoietic Reconstitution
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Gating strategies can be found in Supplemental Information (Figures S7 and S8 ). For analysis of mixed chimerism, cells were first stained with LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit (ThermoFisher Scientific) and blocked with TruStain FcX anti-mouse (Biolegend) for 10 min on ice in Cell Stain Buffer (Biolegend). Antibodies used for staining from Biolegend were as follows: CD45.1 PerCp-Cy5.5 (A20), CD45.2 Pacific Blue (104), CD3 AF488 (17A2), CD4 PE (RM4–4), CD11b BV605 (M1/70), CD19 PE-Cy7 (6D5), CD49b APC (DX5), CD25 PE-Cy5 (PC61), CD8a PE (53–6.7), Ter-119 PE, CD11b PE (M1/70), Gr-1 PE (RB6–8C5), CD3 PE (17A2), B220 PE (RA3–6B2), CD317 PE (927), CD172a APC (P84), CD11c AF700 (N418), CD304 PE (3E12), FOXP3 AF647 (150D), Helios AF488 (22F6), B220 FITC (RA3–6B2), CD8a BV510 (53–6.7). eBioscience antibodies used were as follows: CD117 APC (2B8), Sca-1 Pe-Cy7 (D7). Staining of intracellular markers was conducted with Biolegend True-Nuclear Transcription Factor Buffer Set as per manufacturer’s instructions. For live cell sorting, propidium iodide (MilliporeSigma) was used to determine viability. Cells were analyzed and/or sorted with a BD FACSAria II. Data were analyzed using FlowJo (10.7).
9
Murine Immune Cell Profiling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All antibodies utilized were anti-mouse. CD45.2-Pacific Blue, CD45.2-Alexa Fluor 700, CD11b-PE/Cy7, F4/80-PE/Dazzle594, Ly6C-PE, and Ly6G-PE/Cy7were purchased from Biolegend (San Diego, CA, USA). CD11b PerCP-Cy5.5 was purchased from BD Biosciences (San Jose, CA, USA). MPO-FITC was purchased from LSBio (Seattle, WA, USA). TMEM119-Alexa Fluor 647 was purchased from Abcam (Cambridge, England, UK). The anti-CD16/CD32 2.4G2 mAb was grown locally and used as a mouse Fc block. All immune cell isolations were performed using polypropylene plasticware and the cell suspensions were kept on ice whenever possible.
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!