Krebs ringer hepes buffer

At 13 weeks, male rats were weighed and euthanized by CO₂ asphyxiation in the morning hours. Spleens were collected aseptically, weighed, and immune cells were isolated for further processing. Isolation of immune cells from the spleen have been previously described (27 (link)). Briefly, single cell suspensions were obtained by disrupting tissue through a nylon mesh screen in sterile Krebs-Ringer HEPES buffer with bovine serum albumin (5 g/l; Sigma-Aldrich Canada Ltd., Oakville, ON, Canada). Ammonium chloride lysis buffer (155 mM NH4Cl, 0.1 mM EDTA, 10 mM KHCO3; Fisher Scientific, Edmonton, AB, Canada) was used to lyse erythrocytes. Cells were washed then re-suspended in complete culture medium (RPMI 1640 media; Life Technologies, Burlington, ON, Canada), supplemented with 5% (v/v) heat-inactivated fetal calf serum, 25 mM HEPES, 2.5 mM 2-mercaptoethanol and 1% antibiotic/antimycotic (pH 7.4; Fisher Scientific, see above). Prior to ex vivo analyses, a haemocytometer was used to count live cells using trypan blue dye exclusion (Sigma-Aldrich, as above) to assess cell viability and was >90% for all treatment groups. All cell suspensions were then diluted to 1.25 × 10⁶ cells/ml.

At 13 weeks, male rats were weighed and euthanized by CO₂ asphyxiation in the morning hours. Spleens were collected aseptically, weighed, and immune cells were isolated for further processing. Isolation of immune cells from the spleen have been previously described (27 (link)). Briefly, single cell suspensions were obtained by disrupting tissue through a nylon mesh screen in sterile Krebs-Ringer HEPES buffer with bovine serum albumin (5 g/l; Sigma-Aldrich Canada Ltd., Oakville, ON, Canada). Ammonium chloride lysis buffer (155 mM NH4Cl, 0.1 mM EDTA, 10 mM KHCO3; Fisher Scientific, Edmonton, AB, Canada) was used to lyse erythrocytes. Cells were washed then re-suspended in complete culture medium (RPMI 1640 media; Life Technologies, Burlington, ON, Canada), supplemented with 5% (v/v) heat-inactivated fetal calf serum, 25 mM HEPES, 2.5 mM 2-mercaptoethanol and 1% antibiotic/antimycotic (pH 7.4; Fisher Scientific, see above). Prior to ex vivo analyses, a haemocytometer was used to count live cells using trypan blue dye exclusion (Sigma-Aldrich, as above) to assess cell viability and was >90% for all treatment groups. All cell suspensions were then diluted to 1.25 × 10⁶ cells/ml.