preview

Heterophile Isolation Lab Report

Decent Essays

Heterophile isolation
Polymorphonuclear heterophils were isolated from normal chicken using a modification of previously described procedures(Andreasen et al., 1991; Brooks et al., 1996). In brife ,whole venous blood was collected in sodium citrate (0.129 M; pH 6.5; 9:1,v/v). The blood was diluted in RPMI 1640 (1:1) on a Ficoll-Hypaq gradient . After centrifugation at 400 g for 30 minutes, the plasma and the mononuclear cell layer were removed, and contaminant erythrocytes were removed by hypotonic lysis. Hetrophils were counted in a Neuber chamber, and the viability of the cells was determined by Trypan blue dye exclusion. Purity of live neutrophils was 94% following this procedure. Phagocytosis and killing of opsonized bacteria by heterophils
Phagocytosis and killing of opsonized bacteri were checked by a colorimetric assay according to previously described procedures with some modifications (Fijalkowski et al., 2012; Mehrzad et al., 2009). This experiments wre run in triplicate for each isolated E coli. Brifly, each isolated E coli was opsonized with 10% pooled avian serum for 30 min. The test tube (T) was containe the same volume (500 µL) of heterophiles and opsonized E coli. Test tube was rotated end-over-end at 37 °C for 90 min . The ratio of E coli to hetrophil was 10/1.Control samples (C) contained opsonized E coli, without hetrophil. A 100 µL …show more content…

The heterophiles (1 × 106 cell/mL) were mixed with 0.1% NBT in PBS (pH 7.4) and heat-killed opsonized E coli (1 × 106 cell/mL). The mixture was incubated for 30 min at 37 oC. The unused NBT was, then, removed through washing and the reduced dye was extracted in KOH (2 M) and DMSO and quantitated at 520 nm. The assay was run in triplicate for each isolated E

Get Access