Casy tt cell counter

BDNF secretion was quantified in cultures of control and BDNF-transfected cells after incubation for a defined time in a defined volume of medium. Supernatants were analyzed using the Human Free BDNF Quantikine ELISA Kit (R&D Systems, Minneapolis, MN) according to the manufacturer’s protocol. For quantification as a function of the cell number, cells were trypsinized with 0.05% trypsin-EDTA and counted. Counting of ARPE-19 cells was performed using the CASY Cell Counter TT (Roche, Basel, Switzerland). Primary hRPE cells were mixed with trypan blue solution and counted using a hemocytometer.

For mouse lymphocyte staining, single-cell suspensions from iLNs were prepared by mechanical disruption of the tissues through a 70-µm mesh in 2% FBS in PBS. For stromal cell staining, single-cell suspensions from iLNs were prepared by enzymatic digestion with 0.2 mg ml⁻¹ Collagenase P (no. 11213865001, Sigma), 0.8 mg ml⁻¹ Dispase II (no. 4942078001, Sigma) and 0.1 mg ml⁻¹ DNase I (no. 10104159001, Sigma) in plain RPMI medium (no. 11875093 Gibco). The cell number and viability of samples were acquired using a CASY TT Cell Counter (Roche). For both mouse and human work, cells were stained with surface antibody stains (Supplementary Tables 1 and 2) for 30 min to 2 h at 4 °C in Brilliant stain buffer (no. 563794, BD Biosciences), then washed with 2% FBS in PBS and fixed using the Foxp3/Transcription Factor Staining Buffer Set (no. 00-5323-00, eBioscience). For intracellular staining, cells were incubated for 1 h at 4 °C with the appropriate antibodies. Samples were acquired on an LSR Fortessa (BD Biosciences) using BD FACSDiva software v.9.0 or on a Cytek Aurora Spectral Cytometer (Cytek) using SpectroFlo Software v.3.0, and analysis was done using FlowJo v.10 software (Tree Star).