Difficult to Get Good Staining

Immunology
Columbia University
Graduate Student

Overall

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Company:

BioLegend

Product Name:

PE anti-HMGB1 Antibody

Catalog Number:

651403

Clone Number:

3E8

We used HMBG1 to assess the levels of stress and possible mechanism of cell death of cells exposed to hypoxia. We stained both intracellularly and extracellularly as well as in combination to see which one would yield the best results. The intracellular stain resulted in the greatest shift in MFI, with additional extracellular stain not contributing any more intensity. All in all, HMGB1 seems to be very difficult to assess as it may be released from the cell rather than expressed on or in it.

Experimental Design and Results Summary

Applications

Flow Cytometry

Sample

DU145 Cell Line

Primary Incubation

ECS for 20 mins at RT

Blocking Agent

N/A

Secondary Incubation

Fix/Perm at RT for 40 mins

Tertiary Incubation

ICS at RT for 30 mins 1:50

Detection

Flow Cytometry

Results Summary

We incubated DU145 human cells that had been exposed to hypoxic conditions with HMGB1 antibody as an intracellular stain after the primary extracellular stain and fixation/permeabilization. HMGB1 expression seemed to be slightly elevated and two populations could be seen. However, it was difficult to find a good positive control to corroborate the trends we saw. We tried using UV damage, bleach, ethanol, serum starvation, and DMSO to engage cell death processes, but none of them seemed to increase the intensity of HMGB1 staining.

DOI or PMID #

N/A

Additional Notes

N/A

Related Categories

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Summary

The Good

It seems to stain

The Bad

Stain is very dim, difficult to get positive control

The Bottom Line

Not sure if this is actually staining HMGB1, since none of the death controls seemed to elevate the MFI of HMGB1 in a significant way. However, it does seem that hypoxia may slightly elevate the expression of HMGB1. This antibody was a little difficult to validate.

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