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At each time point, non attached planktonic bacteria were removed and the plate was rinsed thoroughly.
Non attached cells were gently removed with PBS, and adherent PACs were fixed with 4% paraformaldehyde and counted in 10 randomly selected field.
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To distinguish between attached and non-attached PBMCs, the chamber was subsequently superfused with Hank's buffer containing 0.5%% bovine serum albumin.
Non-attached cells were then removed, and the attached macrophages and neutrophils were supplied with fresh L-15 medium and incubated at 30°C for an additional 3 hours before confocal imaging.
In colon, the mucus forms two layers; an outer non-attached and an inner mucus layer that is firmly attached to the epithelium 14.
The cells were allowed to attach for 4 5 hours, thereafter the medium and the non-attached cells were aspirated and fresh medium added for culture over night.
Cells were counted before replating and non-attached cells are not included in the calculation because the cells were allowed to attach for only 10 or 20 min.
After extensive washing to remove non-attached bacteria, an aliquot was maintained on ice to be used for bacterial attachment control.
Tuesday's motion was supported by 44 from the Ukip-led caucus and 32 non-attached MEPs who included Marine Le Pen, the leader of France's National Front.
The 11 Ukip party members will all vote no, joining forces with 42 Greens, 41 Nationalists and several non-attached MEPs.
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CEO of Professional Science Editing for Scientists @ prosciediting.com