Enzymatic amplification staining for flow cytometric analysis of cell surface molecules

CD4+ and CD8+ T Cells Kill IntracellularMycobacterium tuberculosisby a Perforin and Fas/Fas Ligand-Independent Mechanism

“…In fact, up-regulation of CD95L was detected on MTB-activated T cells from only one of four donors using a novel highly sensitive flow cytometry enhancement technique that overcomes sensitivity limitations for detection of CD95L (data not shown; Ref. 48). Furthermore, Oddo et al (49) found that MTB infection of macrophages caused down-regulation of CD95 on the cell surface, which also could explain why there was only a small contribution by the CD95-CD95L pathway to cytolysis of infected MN.…”

Glucocorticoids upregulate CD40 ligand expression and induce CD40L-dependent immunoglobulin isotype switching

“…Cells were cultured for 6 hours with medium, PMA (20 ng/ml; Sigma Chemical Co.) and IO (0.5 µM; Calbiochem-Novabiochem Corp., San Diego, California, USA), or HC (10 -6 M), The cells were then harvested, washed in PBS containing 2% BSA, 5% FCS, and 0.005% sodium azide, and resuspended in 50 µl of this buffer supplemented with human IgG (300 µg/ml) to block nonspecific binding. Staining of cells was performed either by the standard method or by the enzymatic amplification technique developed by Kaplan and Smith (29). In the standard method, the cells were stained with either anti-CD40L phycoerythrin (PE) mAb or isotype control (Ancell Corp., Bayport, Minnesota, USA), according to the manufacturer's instructions.…”

Down-regulation of the forkhead transcription factor Foxp1 is required for monocyte differentiation and macrophage function

“…Western blot analysis demonstrated reduced expression of both the 160-kDa and 135-kDa forms of M-CSFR in monocyte lysates from macFoxp1tg compared with wild-type mice ( Figure 6B). Surface expression of M-CSFR was also investigated by flow cytometry using 2-color staining for monocytes (PE-conjugated MOMA-2) and M-CSFR using FITC enzymatic amplification staining 30 ( Figure 6C). The fraction of MOMA-2-positive cells expressing M-CSFR was significantly lower in macFOXP1tg compared with wild-type mice (percentage of double-positive cells: 4.9 Ϯ 1.6 vs 10.1 Ϯ 1.9%, n ϭ 5 per genotype; P ϭ .002).…”