HT22 Cell Culture

Cells were cultured in Dulbecco's Modified Eagle Medium (DMEM), supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin to prevent microbial contamination. The culture was maintained in a humidified incubator at 37 °C with an atmosphere of 5% CO2. HT22 cells were passaged upon reaching an approximate confluence of 80–90%. For passaging, cells were detached using 0.25% trypsin–EDTA solution, neutralized in complete medium, and seeded at a density of 5,000 to 10,000 cells/cm2, depending on the experimental setup. Care was taken to use cells at low passage numbers to maintain their physiological relevance.

Preparation of HT22 Cells Overexpressing VGLUT1 via rLV Infection for Gene Transfer Efficiency Analysis

In order to determine the more efficient viral vector for gene transfert expression in target cells, GIGA Viral Vectors platform (University of Liège, Liège, Belgium) tested different ready-to use lentiviral vectors (MOI = 50) that should allow fluorescent protein expression under the control of different promoters (hPGK, SV40, mPGK, CBh, EF1a, CMV, SFFV CAG) and having different envelope (VSV-G, Measles, RD114 and GalV). SFFV promoter combine with VSV-g pseudotyped LV was the most efficient combination. Knowing that, gene transfer lentiviral plasmids were purchased from Vector Builder, pLV SFFV VGlu1-IRES-mCherry (VB220831-1419dpk) for and pLV SFFV luc2-IRES-mCherry (CMV Puro) as control (VB220901-1439asa). The plasmid allows expression of VGlu1 (or mSlc17a7[NM_182993.2]) and mCherry. Lentiviral vectors were generated by GIGA Viral Vectors platform (University of Liège, Liège, Belgium). Lenti-X 293 T cells (Clontech Laboratories, Inc., Mountain View, CA, USA) were co-transfected with gene transfer lentiviral plasmids, pSPAX2 (Addgene, Cambridge, MA, USA) and VSV-G encoding vectors (Cell Biolabs, Inc., San Diego, CA, USA). Lentiviral supernatants were collected at 96 h post-transfection, filtrated, concentrated with lenti-Pac Lentivirus Concentration Solution (BioCat #LT007-GC), titrated and used to transduce HT22 cells (MOI = 50) in order to allow the expression of VGlu1 and mCherry (or Luciferase as control). Transduced cells were selected with 1 µg/mL puromycin. The absence of RCL and mycoplasma in cell supernatant was confirmed with qPCR Lentivirus Titration kit (Lonza, Basel, Switzerland) and MycoAlert PLUS Mycoplasma Detection Kit (Lonza, Basel, Switzerland), respectively. The effectiveness of vectors used on the expression level of VGLUT1 protein has been verified using immunofluorescent staining and imaging with confocal microscopy as described below (Fig. 1).

Fig. 1

The immunofluorescent staining shows VGLUT1 expression in wild-type (WT) HT22 cell cultures (left), the same cell line transduced with a blank vector (middle), and HT22 cells transduced with an rLV vector containing the VGLUT1 expression construct (right). The scale bar = 50 μm

Oxygen Glucose Deprivation (OGD)

The experiment was conducted using HT22 immortalized hippocampal neurons cell lines transduced with rLV SFFV-Vglut1-IRES-mCherry to gain VGLUT1 overexpression or rLV SFFV-luc2-IRES-mCherry as a control construct. Cells were seeded into 96-well sterile test plates in standard culture medium (10% FBS in DMEM). Cellular suspension used for the experiment was 3000 cells/well. After 24 h of incubation, the medium was changed to FBS-depleted medium (1% FBS in DMEM). CSB6B solutions were added to selected wells at final concentrations: 0.1, 1.0 and 10.0 µM, 2 h prior to OGD. After this period of time medium was replaced with fresh glucose-free, FBS-depleted medium and CSB6B solutions were added at the concentrations described above, into respective wells. HT22 cells were then subjected to OGD in a hypoxic chamber in an incubator (5%CO2/95%N2) at 37 °C for 18 h. Cells were next transferred to normoxia conditions and medium was replaced with standard culture medium. After 3-h incubation period cells were subjected to viability and cytotoxicity tests as well as immunofluorescence staining and mitochondria membrane potential assay.

Immunofluorescence Staining

Three hours after reoxygenation, culture medium was removed, and cells were washed with PBS. Cells were next fixed with freshly prepared 4% paraformaldehyde (PFA) aqueous solution for 15 min at room temperature (RT). Cells were rinsed with PBS and permeabilized with 0.3% Triton-X in PBS for 15 min at RT. Cells were washed again with PBS and incubated in 5% normal goat serum in PBS for 1 h at RT. Next, cells were incubated with specific primary antibodies solutions at 4 °C overnight. The following day cells were washed with PBS and incubated with specific secondary antibody solution for 1 h at RT in the dark (Table 1). After the last washing step in PBS, cells were counter-stained with DAPI mounting solution (Vector). Cells were visualized using Leica Stellaris 8 confocal microscope and Leica LAS X software. The fluorescence intensity was measured using Leica LAS X software. For preprocessing at each image (n = 7 per group) cells were segmented, histogram was set at the same level along the study, and the mean fluorescence was read across all analyzed images. Since the culture density was relatively low—there were no necessity to use any filters, as such the raw results are presented as the relative fluorescence units (RFU).

Table 1 Antibodies used in immunofluorescent studyCell Viability

Viability of HT22 cells after the indicated treatments was evaluated using the resazurin-based PrestoBlue™ reagent (Invitrogen, ThermoFischer Scientific, USA). Briefly, the reagent was warmed to room temperature before use and then 10 µl of PrestoBlue solution was added to cells in 90 µl culture medium in each well. The plates were incubated for 30 min at 37 °C (5% CO2), protected from light. The absorbance of resorufin, a red compound formed in the reducing environment of living cells, was measured using an Infinite M200 Pro plate reader (Tecan, Switzerland) at 570 nm, with 630 nm as reference wavelength. The cell viability was corrected for background absorbance (wells with cell culture medium only) and expressed as a percentage relative to control.

LDH Release Assay

The extracellular lactate dehydrogenase (LDH) was measured using a CyQUANT™ LDH Cytotoxicity Assay Kit (Invitrogen, ThermoFisher Scientific, USA), following the manufacturer’s protocol. Combined cell culture media aspirated after OGD and after reoxygenation time were incubated with the reagent mixture at RT for 30 min, protected from light. The level of a red formazan product, which is directly proportional to the amount of LDH released into the medium, was measured immediately after adding stop solution using an Infinite M200 Pro plate reader (Tecan, Switzerland) at 490 nm and 680 nm (background). Triton-X 100-treated cells were used as a positive control. The results were corrected for background absorbance (wells with cell culture medium only) and expressed as a percentage relative to control group.

Measurement of Mitochondrial Membrane Potential

Mitochondrial membrane potential of cells was determined after the indicated treatments using cationic dye JC-1 (Cayman chemical, USA). After removing culture medium, cells were incubated with JC-1 staining solution for 30 min in the incubator, following the manufacturer’s protocol. Afterwards, the cells were washed and fluorescence was quantified by a Fluoroskan Ascent FL plate reader (Thermo Fisher Scientific, USA) as well as visualized using a Stellaris 8 WLL DLS confocal microscope (Leica, Germany). Red fluorescence (with excitation and emission at 535 nm and 595 nm wavelength, respectively) was proportional to the number of healthy cells, while green fluorescence (with excitation and emission at 485 nm and 535 nm, respectively) to cells with low mitochondrial membrane potential. The ratio of green to red fluorescence signal was calculated and the results were expressed as a ratio.

Statistics

All data are expressed as the mean ± standard deviation (SD). All data were analyzed using one-way ANOVA. If statistical significance was found after ANOVA, Sidak’s post hoc test was conducted to test the comparisons between experimental groups. A calculated p value < 0.05 was considered statistically significant. Calculations were performed using GraphPad Prism version 10.2.1 for macOS, GraphPad Software, Boston, Massachusetts USA, www.graphpad.com.