Epithelial Antigen, Ber-EP4, Unconjugated

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	The cells were concentrated by centrifugation for 240 seconds at 1200 g and after discarding the supernatant, 100 microliters (μL) of the cell suspension were stained using 5 μL of CD326 (clone Ber‐Ep4, DAKO, Denmark) conjugated with fluorescein isothiocyanate (FITC)

Protocol tips
The cells were concentrated by centrifugation for 240 seconds at 1200 g and after discarding the supernatant, 100 microliters (μL) of the cell suspension were stained using 5 μL of CD326 (clone Ber‐Ep4, DAKO, Denmark) conjugated with fluorescein isothiocyanate (FITC)

Publication protocol

"In the case of FNAs and lymph node biopsies or other tissues, mechanical disaggregation was obtained using a Medimachine™. The rest of the samples already contained the cells in suspension. The cells were concentrated by centrifugation for 240 seconds at 1200 g and after discarding the supernatant, 100 microliters (μL) of the cell suspension were stained using 5 μL of CD326 (clone Ber‐Ep4, DAKO, Denmark) conjugated with fluorescein isothiocyanate (FITC) and 5 μL of CD33 (Clone P67.6, Becton Dickinson) conjugated with phycoerythrin (PE). We have tested two different clones of CD45, (one of them in two different fluorocromes): 5 μL of clone HI30 (Invitrogen) conjugated with pacific orange (PO), 5 μL of clone 2D1 (Becton Dickinson) conjugated with peridinin chlorophyllic protein cyanine 5.5 (PerCP‐Cy5.5), and 2 μL of clone 2D1 (Becton Dickinson) conjugated with allophycocyanin (APC). Reagents were incubated with the cells in the dark at room temperature for 15 minutes. The cells were then lysed for 10 minutes with 2 mililiters (mL) of FacsLyse (Becton Dickinson) and centrifuged for 240 seconds at 1200 g. The supernatant was aspirated and discarded. The cells were then washed with 2 mL of CellWash (Becton Dickinson), ressuspended with 500 μL of FacsFlow (Becton Dickinson) and acquired in a FacsCalibur or FACSCanto II flow cytometer (Becton‐Dickinson, San Jose, CA) with Cell‐quest or BD FacsDiva softwares. For each measurement up to 100,000 events were acquired.

A fluorescence minus one (FMO) control 17 including CD45 and CD33 was used in every sample to validate the positivity for CD326. (Fig. 1)
Analysis was performed using Paint‐a‐gate™ or Infinicyt™ softwares.

As illustrated in Figure 1, leukocytes were identified by their expression of anti‐CD45 vs. SSC. Among the CD45 positive population, the group with the lowest internal complexity, negative for CD33 corresponded to the lymphocytes, and the rest of the CD45 and CD33 positive cells corresponded to the granulocytes (dim co‐expression) and the monocytes/macrophages (strong co‐expression). Light scatter was used to exclude debris (FSC vs.SSC) and doublets (FSC‐Area vs. FSC‐Height).

Large cells were identified as metastasis when they were CD45 negative, CD33 negative and strongly CD326 positive (Fig. 2). The differential diagnosis between reactive mesothelial cells and metastatic ACA is often extremely difficult in serous effusion specimens. To discriminate between mesothelial cells and malignant epithelial cells, which are both CD45 and CD33 negative, the expression of CD326 is crucial. Mesothelial cells are negative and malignant epithelial cells usually exhibit a strong expression, with at least a difference of one mean fluorescence intensity (MFI) decade (Fig. 3)."

Full paper   Login or join for free to view the full paper.

Reviews

Discussion

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing Flow cytometry Anti-bodies Human - CD326/EpCAM using Epithelial Antigen, Ber-EP4, Unconjugated from Agilent Technologies.

Manufacturer protocol

Download the product protocol from Agilent Technologies for Epithelial Antigen, Ber-EP4, Unconjugated below.

Download manufacturer protocol

Videos

Check out videos that might be relevant for performing Flow cytometry Anti-bodies Human - CD326/EpCAM using Epithelial Antigen, Ber-EP4, Unconjugated from Agilent Technologies. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.