ioGlutamatergic Neurons are compatible with plates ranging from 6 to 384 wells and are available in two vial sizes, tailored to suit your experimental needs with minimal waste.  

-  Recommended seeding density for ioGlutamatergic Neurons is 30,000 cells/cm², compared to up to 250,000 cells/cm² for other available products on the market.
-  One Small vial (0.75 x 10⁶ viable cells) can plate a minimum of 0.5 x 24-well plate, 0.75 x 96-well plate, or 1 x 384-well plate.
-  One Large vial (1.5 x 10⁶ viable cells) you can plate a minimum of 1 x 24-well plate, 1.5 x 96-well plate, or 2 x 384-well plates.

Immunocytochemistry of ioGlutamatergic Neurons at day 9 post plating shows homogeneous expression of pan neuronal marker MAP2 and a dense neuronal network. Image courtesy of Charles River Laboratories.

Immunofluorescent staining on post-revival day 11 demonstrates homogenous expression of pan-neuronal proteins (MAP2 and TUBB3) and glutamatergic neuron-specific transporters (VGLUT1 and VGLUT2). Cells exhibit neurite outgrowth.

Immunofluorescence staining of NeuN using ab177487 in ioGlutamatergic Neurons (Human iPSC-Derived Glutamatergic Neurons, ab259259), which were differentiated for 1 day post induction.

The cells were fixed with 4% formaldehyde (10 min), permeabilized with 0.1% PBS-Tween for 5 mins and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab177487 at 1 μg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin, at 1/1000 dilution. Cells were then incubated with ab150081, Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 dilution (shown in green) and ab150120, Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) preadsorbed at 1/1000 dilution (shown in red). Nuclear DNA was labelled with DAPI (shown in blue).

Images were acquired with the Perkin Elmer Operetta HCA and a maximum intensity projection of confocal sections is shown.

Immunofluorescence staining of beta III Tubulin using ab52623 in ioGlutamatergic Neurons (Human iPSC-Derived Glutamatergic Neurons, ab259259), which were differentiated for 11 days post induction.

The cells were fixed with 4% formaldehyde (10 min), permeabilized with 0.1% PBS-Tween for 5 mins and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab52623 at 0.1 μg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin, at 1/1000 dilution. Cells were then incubated with ab150081, Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 dilution (shown in green) and ab150120, Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) preadsorbed at 1/1000 dilution (shown in red). Nuclear DNA was labelled with DAPI (shown in blue).

Images were acquired with the Perkin Elmer Operetta HCA and a maximum intensity projection of confocal sections is shown.

Immunofluorescence staining of MAP2 using ab183830 in ioGlutamatergic Neurons (Human iPSC-Derived Glutamatergic Neurons, ab259259), which were differentiated for 11 days post induction.

The cells were fixed with 4% formaldehyde (10 min), permeabilized with 0.1% PBS-Tween for 5 mins and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab183830 at 1 μg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin, at 1/1000 dilution. Cells were then incubated with ab150081, Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 dilution (shown in green) and ab150120, Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) preadsorbed at 1/1000 dilution (shown in red). Nuclear DNA was labelled with DAPI (shown in blue).

Images were acquired with the Perkin Elmer Operetta HCA and a maximum intensity projection of confocal sections is shown.

Immunofluorescence staining of Tau using ab76128 in ioGlutamatergic Neurons (Human iPSC-Derived Glutamatergic Neurons, ab259259), which were differentiated for 11 days post induction.

The cells were fixed with 4% formaldehyde (10 min), permeabilized with 0.1% PBS-Tween for 5 mins and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab76128 at 0.5 μg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin, at 1/1000 dilution. Cells were then incubated with ab150081, Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 dilution (shown in green) and ab150120, Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) preadsorbed at 1/1000 dilution (shown in red). Nuclear DNA was labelled with DAPI (shown in blue).

Images were acquired with the Perkin Elmer Operetta HCA and a maximum intensity projection of confocal sections is shown.

ioGlutamatergic Neurons after revival over the course of the first 11 days of culture. Day 1 to 11 post-thawing; 400X magnification; scale bar: 100µm

ioGlutamatergic Neurons display neuronal activity that matures over-time. Examples of MaxOne high-resolution multi electrode array (MEA) recordings of ioGlutamatergic Neurons in BrainPhys™ media. The activity maps show firing rate (A), spike amplitude (B) and % of active electrodes (C). Results demonstrate a time-dependent increase of spontaneous activity during neuronal maturation from 2 to 3 weeks post-revival. Iovino et al., Molecular and functional characterization of stem cells-derived glutamatergic neurons ioGlutamatergic Neurons in support of drug discovery applications., Charles River Laboratories.

ioGlutamatergic Neurons show good suitability for high-throughput screening in 384-well format plates. Cytotoxicity CellTiter-Glo^®️ (CTG) and TR-FRET (HTRF^®️) assays for AKT serine/threonine kinase 1 (AKT) and Huntingtin (HTT) proteins were performed on ioGlutamatergic Neurons in 384-well plates treated with tool compound (cmp) at day 9 post-revival. Compound titration results in a concentration response curve for all three assays (mean±sd of 2 replicates). CTG assay on ioGlutamatergic Neurons shows an excellent average signal/ background ratio and high suitability for HTS. HTRF^®assays on ioGlutamatergic Neurons show lower signals but with low variability, and could therefore also provide a suitable platform for HTS. Iovino et al., Molecular and functional characterization of stem cells-derived glutamatergic neurons ioGlutamatergic Neurons in support of drug discovery applications., Charles River Laboratories.

ioGlutamatergic Neurons are delivered in a cryopreserved format and are programmed to rapidly mature upon revival in the recommended media. The protocol for the generation of these cells is a three-phase process:
1. Induction (carried out at bit.bio)
2. Stabilization for 4 days with Doxycycline
3. Maintenance during which the neurons mature.