Atplite luminescence assay kit

The migration of splenic and BM B cell populations in response to chemokine gradients was performed in 6-well Costar transwell chambers (5 μm pore size, Corning). Briefly, a suspension of 10⁶ mononuclear cells in RPMI 1640 supplemented with 10% FCS was added to each insert in a well containing a solution of chemokine (R&D Systems). In some experiments, blocking anti-CXCR4 MAB21651 (247506, R&D Systems) or anti-CCR7 MAB3477 (4B12, R&D Systems) antibodies were added to the cells prior to migration. Wells containing medium without chemokines were used as controls. After 2 h at 37°C, cells in the bottom of the wells were harvested, diluted in FACS staining buffer and incubated with antibodies and counting beads to discriminate and quantify B cell subsets. The migration of Nalm-6 cells was performed in 96-well Costar transwell chambers (5 μm pore size, Corning NY). A cell suspension of 10⁴ Nalm-6 in RPMI 1640 supplemented with 10% FCS was added to each insert. Wells containing medium without chemokines were used as controls. After 1 h at 37°C, cells in the bottom of the wells were counted by using the ATPlite luminescence assay kit (PerkinElmer, Waltham, MA). The results are expressed as chemotaxis index, i.e., the ratio of cells migrating in response to the chemoattractant over cells migrating toward the medium alone.

Cybrid cells were seeded in a 96 well plate at 1 × 10³ cells/well with growth medium containing 25 mM glucose, and incubated for 24 h. The ATP content was measured using an ATPlite luminescence assay kit (Perkin Elmer) 24 h after cell culture in medium containing 25 mM galactose or 25 mM glucose. Protein concentration was determined by the bicinchoninic acid assay (Pierce™ BCA Protein Assay Kit, Thermo Fisher Scientific). The values for ATP content were normalized to the mitochondrial protein concentration.

White 96-well cell culture plates were used, and cells were seeded at a density of 5 × 10⁵ cells/well. To determine synthesized ATP contents, the ATPlite luminescence assay kit (Cat. No: 6016943) from Perkin Elmer (Billerica, MA, USA) was employed according to the manufacturer’s instructions.

Short-term adhesion assays were performed as previously described (8 (link)). Briefly, 2.10⁴ Nalm-6 cells in suspension in PBS supplemented with 0.1% BSA were stimulated with 300 nM CXCL12 for 1 min and then added to VCAM-1-coated wells (with a 1 mg/ml solution). Cells were quickly spun down and allowed to settle for another minute at 37°C. As controls, cells unstimulated with CXCL12 and uncoated wells were used. Wells were then washed twice and the number of adherent cells was determined using the ATPlite luminescence assay kit (PerkinElmer, Waltham, MA).

Cells were treated with various concentrations of HSR for 1 h before the addition of 0.25 mM corticosterone. The total cellular ATP content was determined using an ATPlite luminescence assay kit (PerkinElmer, Waltham, MA, USA) and a TriStar LB 941 multimode microplate reader (Berthold Technologies, Calmbacher, Germany). The ATP content was determined using an internal standard and expressed as a percentage of the ATP content in untreated cells (control).