The pancreas was collected from the adult p19Arf−/− mice and transferred to a 15 ml tube containing 5 ml of cold collagenase P (Roche, San Francisco, CA, USA). Following incubation for 18–20 min in a 37 °C water bath, the tissues were washed with 5 ml of quenching buffer consisting of HBSS with 10% FBS and isolated by centrifugation at 300 × g for 3 min at room temperature. The tissues were washed with another 10 ml of cold quenching buffer, centrifuged at 300 g for 3 min, and incubated with 2 ml of 25% Ficoll (Sigma-Aldrich, St Louis, MO, USA; F8016). After addition to a Ficoll gradient of 23, 21 and 13%, the tissues were centrifuged at 2500 g for 20 min at 4 °C. The islet layer was visible between the 13 and 23% gradients. The islets were collected and washed with 10 ml of regular cell culture media, centrifuged at 1000 g for 5 min, and resuspended in 1 ml of RPMI 1640-complete medium (Biowest LLC, Kansas City, MO, USA).
Rpmi 1640 complete medium
Manufactured by Biowest
Sourced in United States
RPMI 1640-complete medium is a cell culture medium formulated to support the growth and maintenance of a variety of cell types. It provides the necessary nutrients, vitamins, and other components required for cell proliferation and survival in an in vitro environment.
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Lab products found in correlation
2 protocols using rpmi 1640 complete medium
1
Pancreatic Islet Isolation from p19Arf−/− Mice
2
Ex vivo IFN-γ Production in Canine Leishmaniasis
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A total of 69 dogs were conveniently selected for determination of ex vivo IFN-γ specific concentrations after L. infantum or rKMP11 stimulation of whole blood (Group 1; n = 25, Group 2; n = 15, Group 3; n = 16 and Group 4, n = 13) as previously described [13 (link),31 (link)]. Briefly, five hundred μL of heparinized blood was diluted to a ratio of 1:10 with RPMI 1640 complete medium (Biowest, Nuaillé, Frances) per each well and incubated in 12-well flat bottom plastic culture Costar® plates 3596 (Corning, Corning, NY, USA). Experimental treatments were as follows: (1) medium alone, (2) LSA at 10 μg/mL, (3) recombinant kinetoplastic protein 11 (rKMP11) at 10 μg/mL and (4) mitogen ConA (100 mg, Medicago, Uppsala, Sweden) at of 10 μg/mL. All treatment conditions were incubated for 5 days at 37 °C in 5% of CO2 air. Then, blood was collected and centrifuged at 300× g for 10 min and the supernatant was collected and stored at −80 °C until used.
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