For flow cytometry of tissue culture cells, cells were isolated from mouse spleens and mechanically dissociated with a 40 µm cell strainer (Greiner Bio-One). Red blood cells were lysed with RBC lysing buffer (Sigma). Cells were labeled with aqua live/dead fixable viability dye in PBS (1:500), followed by surface antibodies (1:100) in staining buffer (1% BSA, 1% rat serum in PBS). Antibodies used for surface staining: PE/Dazzle anti-CD4 (RM4-5, Biolegend), APC/CY7 anti-CD8 (YTS156.7.7, Biolegend), PE/CY7 anti-CD44 (IM7, Biolegend), anti-CD69 (H1.2F3, BD Bioscience). For intracellular staining, cells were stained with aqua live/dead dye, followed by surface staining, fixation with perm/fixation solution (Thermofisher), and stained with intracellular antibodies against FITC anti-IFNγ (XMG1.2, Invitrogen) and AF647 anti-tumor necrosis factor α (TNFα) (MP6-XT22, Biolegend) in 1 x perm/wash buffer. Cells were washed and analyzed on a CytoFLEX flow cytometer (Beckman Coulter) and analyzed using FlowJo software.
Anti cd69 h1.2f3
Manufactured by BD
Sourced in Austria, Canada
Anti-CD69 (H1.2F3) is a monoclonal antibody that binds to the CD69 antigen. CD69 is a type II transmembrane glycoprotein expressed on the surface of various hematopoietic cells, including T cells, B cells, and natural killer cells. The core function of this antibody is to detect and bind to the CD69 antigen.
Automatically generated - may contain errors
Lab products found in correlation
Pe cy7 anti cd44 im7, by BioLegend (1 mentions)
Rbc lysing buffer, by Merck Group (1 mentions)
Mp6 xt22, by BioLegend (1 mentions)
40 m cell strainer, by Greiner (1 mentions)
Cytoflex flow cytometer, by Beckman Coulter (1 mentions)
Fluorescence activated cell sorting (facs), by BD (1 mentions)
Anti cd44 im7, by BD (1 mentions)
Anti cd25 pc61, by BD (1 mentions)
Anti cd8α 53 6, by BD (1 mentions)
Anti cd62l mel 14, by Thermo Fisher Scientific (1 mentions)
Live dead fixable near ir, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Lsrfortessa flow cytometer, by BD (1 mentions)
Lsrii system, by BD (1 mentions)
Sytox blue, by Thermo Fisher Scientific (1 mentions)
Diva software, by BD (1 mentions)
Anti cd8a, by BD (1 mentions)
Cyan adp, by Beckman Coulter (1 mentions)
Collagenase 1, by Merck Group (1 mentions)
Lsrii flow cytometer, by BD (1 mentions)
7 protocols using anti cd69 h1.2f3
1
Multiparametric Flow Cytometry of Immune Cells
2
CD8+ T Cell Activation and Migration
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
CD8+ T cells were treated according to the preparative protocol for ACT, as described above. Following the resting period in IL-2/IL-7/IL-15, they were plated at 2 × 105 cells/well in flat 96-well plates and were either unstimulated or treated with CCL2 (100ng/mL) in fluid phase and/or pre-coated anti-CD3e and/or anti-CD28, using clones and concentrations as above. The cells were harvested for analysis after a further 18 hours and stained using anti-CD69 (H1.2F3; BD) and anti-CD25 (BPC61.5; eBiosciences). For the migration assay, Costar (Corning) 24-well plates with 3 μm pore size were used. Transduced T cells were collected, counted and resuspended in IMDM BSA 0,1% at 3 × 106 cells/mL. 100 μL of cells were placed in the upper chamber. The bottom chamber was filled with 500 μL of IMDM BSA 0,1% with or without CCL2 (100 ng/mL). Cells were incubated at 37°C for 2 h to allow migration. Migrated cells were collected from the bottom chambers and counted using FACS (BD).
3
Characterizing OT-I CTL Activation Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Expression of activation‐associated surface molecules on OT‐I CTLs was evaluated on day 3 or 4 post‐transduction or the day that adoptive transfer was performed, which was usually day 6 post‐isolation. Cells were stained with anti‐CD8α (53–6.7, BD Biosciences, San Jose, CA, USA), anti‐CD44 (IM7, BD Biosciences), anti‐CD25 (PC61, BD Biosciences), anti‐CD69 (H1.2F3, BD Biosciences), anti‐CD62L (MEL‐14, eBioscience, Vienna, Austria) and anti‐Vα2 (B20.1, BD Biosciences). Antibodies were used at 1 μg mL−1 in running buffer (5% fetal bovine serum and 2 mm ethylenediaminetetraacetic acid, 0.01% sodium azide in 1 × phosphate‐buffered saline) and the cells were stained for 20 min at 4°C prior to washing two times with running buffer. Cell viability was evaluated using 0.5 μg mL−1 4,6‐diamidino‐2‐phenylindole (Thermo Fisher Scientific) or the LIVE/DEAD Fixable near‐IR (Thermo Fisher Scientific). Data were collected on an LSR Fortessa flow cytometer (BD Biosciences) and analyzed using FlowJo software (TreeStar Inc., Ashland, OR, USA). For fluorescence‐activated cell sorting of medium to high mCherry expressing CTL population, cells were resuspended in FACS buffer (5% fetal calf serum, 2 mm ethylenediaminetetraacetic acid in 1 × phosphate‐buffered saline) and incubated with 4,6‐diamidino‐2‐phenylindole. Cells were either used fresh or cryopreserved according to established methods.85
4
Immunophenotyping of Murine T Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Stained cells were analyzed using an LSRII system (BD Biosciences). Data were analyzed with the Diva software (BD Biosciences). Cell viability was evaluated using SYTOX Blue (Life Technologies). The following antibodies were used: anti‐CD5 (53–7.3), anti‐CD4 (RM4‐5), anti‐CD8a (53–6.7), anti‐TCRb (H57‐597), anti‐CD44 (IM7), and anti‐CD69 (H1.2F3), all from BD Biosciences.
5
Isolation and Flow Cytometric Analysis of Murine Reproductive Tract Immune Cells
6
Multiparameter Flow Cytometry of Engineered T Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The following mAbs and isotype controls were obtained from eBioscience (Hatfield, U.K.): anti-CD8b (H35-17.2), anti-CD4 (GK1.5), anti-Thy1.1 (HIS51), anti-CD45.1 (A20), anti-CD45.2 (104), and anti-Vβ11 (RR3-15). We obtained the following mAbs and isotype controls from BD Pharmingen: anti-CD44 (IM7), anti-CD62L (MEL-14), anti–programmed death 1 (PD-1; J43), and anti-CD69 (H1.2F3). A murine anti–c-myc (9E10, Santa Cruz Biotechnology) and a secondary rat anti-murine IgG Ab (X56, BD Pharmingen) were used to assess TCR α-chain expression. Samples were run on a BD LSRFortessa (BD Biosciences, Oxford, U.K.) and analysis was performed using FlowJo software (Tree Star).
Transduced T cell populations were identified based on a live lymphocyte gate, followed by expression of a congenic marker (where relevant), expression of a coreceptor or coreceptors (CD4 and/or CD8), and then by expression of the c-myc tag (for MDM2- or LoMDM2-TCR–transduced populations) or by expression of Vβ11 (F5-TCR–transduced populations). Where intensity of CD8 expression was investigated, this was excluded from the initial gating strategy, and transduced populations were identified from expression of the relevant congenic marker as well as the introduced TCR.
Transduced T cell populations were identified based on a live lymphocyte gate, followed by expression of a congenic marker (where relevant), expression of a coreceptor or coreceptors (CD4 and/or CD8), and then by expression of the c-myc tag (for MDM2- or LoMDM2-TCR–transduced populations) or by expression of Vβ11 (F5-TCR–transduced populations). Where intensity of CD8 expression was investigated, this was excluded from the initial gating strategy, and transduced populations were identified from expression of the relevant congenic marker as well as the introduced TCR.
7
Fetal Thymus Cell Immunophenotyping
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Anti-CD4 (RM4-5), anti-CD8 (53-6.7) and anti-CD69 (H1.2F3) antibodies were bought from BD Biosciences (Mississauga, ON, Canada). Anti-TCR-b (H57-597) and anti-CD5 (53-7.3) antibodies were purchased from Biolegend (San Diego, CA).
Thymi from E18.5 embryos were dissociated and cell staining was performed as previously described 23 followed by acquisition on a FACSCanto (BD Biosciences). Data analysis was performed using FLOWJO (TreeStar, Ashland, OR).
Thymi from E18.5 embryos were dissociated and cell staining was performed as previously described 23 followed by acquisition on a FACSCanto (BD Biosciences). Data analysis was performed using FLOWJO (TreeStar, Ashland, OR).
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!