To prepare the lung tissues for flow cytometry, they were first cut into small pieces and incubated with 1 mg/ml collagenase D (Worthington) and 0.5 mg/ml DNAse I (Roche) at 37 °C for 30 mins. Collagenase was inactivated with 1 ml sterile DMEM containing 10% FBS; the digested tissues were transferred to a 70-μM nylon cell strainer and disrupted using a syringe plunger to obtain single cell suspensions. Following lung tissue processing, the resulting single cell suspension, BAL, and peripheral blood samples were processed in 1 ml Red Blood Cell Lysing Buffer (Sigma). The cellular populations of the lung tissue, BAL, and peripheral blood from different groups were identified through the following markers: neutrophils were identified with anti-Ly6G (Biolegend) and CD11b (abcom) monoclonal antibody (mAb), macrophages/monocytes were identified with anti-F4/80 (eBiosciencce) and CD11b mAb, T cells were identified with anti-CD4 mAb (BD Pharmingen), eosinophils were identified with anti-siglecF mAb. The stained samples were analyzed with an Attune NxT Acoustic Focusing Cytometer (Thermo Fisher). Dead cells and debris were excluded from analysis via size exclusion.
Anti cd4 mab
Manufactured by BD
Anti-CD4 mAb is a monoclonal antibody that specifically binds to the CD4 receptor on the surface of T cells. This product is intended for use in research applications.
Automatically generated - may contain errors
Lab products found in correlation
Collagenase d, by Worthington (1 mentions)
Attune nxt acoustic focusing cytometer, by Thermo Fisher Scientific (1 mentions)
Anti ly6g, by BioLegend (1 mentions)
Red blood cell lysing buffer, by Merck Group (1 mentions)
Dnase 1, by Roche (1 mentions)
Cd11b, by BD (1 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (1 mentions)
Cd4 t cell isolation kit 2, by Miltenyi Biotec (1 mentions)
Ab15285, by Abcam (1 mentions)
Warp red chromagen, by Biocare Medical (1 mentions)
3 protocols using anti cd4 mab
1
Lung Cell Population Analysis by Flow Cytometry
2
CD4+ T Cell Proliferation Assay with Candida albicans-Pulsed DCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
CD4+ T lymphocytes were isolated from spleen of C57 mice by negative immunomagnetic using a CD4+ T cell isolation kit II (Miltenyi Biotec, Bergisch Gladbach, Germany). Cell purity was assessed by flow cytometry using PE-conjugated anti-CD4 mAbs (eBioscience Inc., San Diego, CA, USA). The purity reached to >95 % in the cell population.
To trace proliferation, T cells were labeled with 10 μm CFSE (Invitrogen, Grand Island, NY, USA) according to the manufacturer’s instructions [28 (link)]. For these assays, spleen CD4+ T cells isolated from 6 of C57 mice were first incubated with 10 μM CFSE (Invitrogen, Carlsbad, CA) at 37 °C for 8 min. Meanwhile, DCs were incubated for 24 h at 37 °C with C. albicans and then washed, co-cultured with the CFSE-labeled allogeneic T cells at different DCs/T cell ratios (0, 1:10, 1:20, 1:40, and 1:80) in RPMI containing 5 % FCS for 2 days at 37 °C in triplicate. After 2 days of incubation, T cell proliferation status was examined by formation of T cell clusters under a microscope and CD4+ T cells were verified by flow cytometric measurement with anti-CD4 mAb (BD Biosciences).
To trace proliferation, T cells were labeled with 10 μm CFSE (Invitrogen, Grand Island, NY, USA) according to the manufacturer’s instructions [28 (link)]. For these assays, spleen CD4+ T cells isolated from 6 of C57 mice were first incubated with 10 μM CFSE (Invitrogen, Carlsbad, CA) at 37 °C for 8 min. Meanwhile, DCs were incubated for 24 h at 37 °C with C. albicans and then washed, co-cultured with the CFSE-labeled allogeneic T cells at different DCs/T cell ratios (0, 1:10, 1:20, 1:40, and 1:80) in RPMI containing 5 % FCS for 2 days at 37 °C in triplicate. After 2 days of incubation, T cell proliferation status was examined by formation of T cell clusters under a microscope and CD4+ T cells were verified by flow cytometric measurement with anti-CD4 mAb (BD Biosciences).
3
Histological Analysis of Rejecting Renal Allografts
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!