Cell Surface Staining Of Human CD28 Receptor From Human PBMC

October 31, 2016

Surgery
George Washington University, Washington DC
Post-doctoral Fellow

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Company:

BioLegend

Product Name:

PE/Cy7 anti-human CD28 Antibody

Catalog Number:

302926

T cell co-stimulatory receptor molecule is important for T cell activation and downstream production of cytokines. CD28 binds to CD80 and CD86 expressed by APC's and this interaction is essential along with TCR  stimulation for T cell activation. An excellent and reliable method to measure surface expression of CD28 on T cell as an index of T cell activation is staining PBMC with fluorochrome conjugated anti-CD3; anti-CD4; anti-CD8 and anti-CD28 human antibodies. PBMCs can be gated on either CD3 (all T cell population) or separately for CD4 or CD8 population and expression of cell surface CD28 measured for evaluation of T cell activation.

Experimental Design and Results Summary

Applications

Flow Cytometry

Sample

Human PBMC or whole blood

Primary Incubation

Cells incubated with PE/Cy7 anti-human CD28 Antibody( 1:100) and FITC anti-human CD3 in a total volume of 100ul for 20-30 mins in dark in 12 x 75-mm tube. After incubation, cells were washed in 2 ml staining buffer. Cells were next centrifuged at 400xg for 5 mins. Supernatant was discarded and cells re-suspended in 500 ul of staining buffer and analyzed in a flow cytometer.

Blocking Agent

Fc receptor block

Secondary Incubation

N/A

Tertiary Incubation

N/A

Detection

Flow cytometric detection of expresssion of cell surface CD28 PEcy7 antibody in Blue-FL-4 channel for the specific cytometer.

Results Summary

T cells were gated by the expression of CD3 surface marker. CD3+ cells were analyzed for expression of CD28. There was a significant fraction of T cells which co-expressed CD28 , depicted by the anti-human CD28 PEcy7 antibody (Blue FL-4 channel) positive fraction. Isotype for mouse IgG1 was used as a negative control to distinguish between CD28+ and CD28- cells.

Additional Notes

Antibody concentration needs standardization. If performing surface staining from whole blood, use 100ul of whole blood and add antibodies in an additional mix of 50ul (final volume 150 ul).

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Summary

The Good

Good and Reliable Method of estimation of surface expression of CD28 in T cells by flow cytometry

The Bad

Nothing to mention

The Bottom Line

Use of Flow cytometric method for detection of expression of co-stimulatory molecule CD28 by T cells by PE/Cy7 anti-human CD28 Antibody is a reliable and inexpensive method for analysis of T cell activation.

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