Dear Sulay Patel!

Here are:

1. Commercially available primary human microglia: Celprogen

http://www.celprogen.com/

2. Human microglia cell line:  https://www.researchgate.net/deref/http%3A%2F%2Fwww.innoprot.com

3. Isolation of Human microglia (method):

Microglial isolation from human brain tissue biopsies

Following surgical resection, tissue was transported to the research facility. Approximately 1–2 g of tissue was washed in HBSS, and meninges and visible blood vessels were removed. Tissue was diced into pieces approximately 1 mm3 using a sterile scalpel and transferred to a 50 mL falcon tube containing 10 mL enzyme dissociation mix (10 U/mL DNase (Invitrogen, CA, USA) and 2.5 U/mL papain (Worthington, NJ, USA) in Hibernate-A medium (Gibco, CA, USA)) per gram of tissue for 10 minutes at 37 °C with gentle rotation. The tissue was removed from the incubator, gently triturated to aid digestion and returned to the incubator for a further 10 minutes. Dissociation was slowed by adding an equal volume of Dulbecco’s modified eagle medium: Nutrient mixture F-12 (DMEM/F12; Gibco, CA, USA) with 1% B27 (Gibco, CA, USA) and the cell suspension was passed through a 70 μm cell strainer (Bector Dickinson, NJ, USA). Cells were centrifuged at 160 × g for 10 minutes, the supernatant discarded and resuspended in 20 mL neural precursor cell (NPC) proliferation media (DMEM/F12 with 1% B27, 1% GlutaMAX (Gibco, CA, USA), 1% penicillin-streptomycin-glutamine (PSG; Gibco, CA, USA), 40 ng/mL fibroblast growth factor-2 (FGF-2; Peprotech, NJ, USA), 40 ng/mL epidermal growth factor (EGF; Peprotech, NJ, USA) and 2 μg/mL heparin (Sigma, MO, USA)). The cell suspension was transferred to a T75 tissue culture flask (Nunc, Roskilde, Denmark) and incubated overnight at 37 °C with 95% air/5% CO2. The following day the tissue culture flask was tapped firmly to remove non-adherent or loosely-adherent cells and these were transferred to a new T75 tissue culture flask. This flask contains predominantly NPC’s, pericytes and astrocytes which can be used as described previously12,20. The original flask containing the adherent cells was washed twice with NPC proliferation media and 15 mL of microglial culture media was added (DMEM/F12 with 10% foetal bovine serum (FBS; Moregate, QLD, Australia) and 1% PSG). Microglia were maintained in this media for up to 1 week at 37 °C with 95% air/5% CO2 with minimal media changes to prevent contaminating cell growth. Once microglia begin to extend processes they can be utilised for further studies. When cultured as described above, microglial yields of 2–300,000 cells/gram of tissue can be expected from epilepsy biopsy samples. GBM biopsies were significantly more varied, but substantially higher than that of epilepsy surgeries. Yields for autopsy samples are also extremely variable and highly dependent on the post-mortem delay. One Parkinson’s disease autopsy sample with a short post mortem delay (2.5 hours) resulted in a yield of ~100,000 cells/gram whilst another Parkinson’s case with a long post mortem delay (17.5 hour hours) produced no viable microglia. An overview of the microglial isolation procedure is summarized in Fig. 1.

Mixed glial isolation from human brain tissue

Mixed glial cultures containing astrocytes, pericytes and microglia were isolated from biopsy adult human brain tissue and cultured for 1–2 weeks before plating as described previously12,21.

Cell plating

To harvest cells for plating, culture media was removed and T75 tissue culture flasks were washed with phosphate buffered saline (PBS). 3 mL of 0.25% trypsin-1 mM ethylenediaminetetraacetic acid (EDTA; Gibco, CA, USA) was added for five minutes at 37 °C with 95% air/5% CO2. Microglia attach firmly to the T75 tissue culture flasks and to aid microglial detachment cells were gently scraped with a rubber cell scraper (Falcon, MA, USA). Trypsin was neutralised by addition of microglia culture media and cells counted using a haemocytometer. Cells were plated at 5,000 cells/well for 96 well plates or 25,000 cells/well for 24 well plates. For confocal experiments cells were plated on glass coverslips inside a 48 well plate at 5,000 cells/well. Cells were allowed to attach overnight before utilisation for experiments.

Immunogen treatment

To investigate microglial inflammatory responses cells were treated for 1–24 hours with 10 ng/mL LPS (from Escherichia coli 026:B6, L4391, Sigma, MO, USA), IFNγ (R&D Systems, MN, USA), IL-1β (Peprotech, NJ, USA) or vehicle (0.1% BSA in PBS).

Immunocytochemistry

Cells were fixed in 4% paraformaldehyde (Scharlau, Spain) for 15 minutes and washed in PBS with 0.1% triton X-100 (PBS-T; Sigma, MO, USA). Cells were incubated overnight with antibodies (Table S1) diluted in immunobuffer (PBS with 0.2% triton X-100, 1% goat serum (Gibco, CA, USA) and 0.04% thimerosal (Sigma, MO, USA)). Cells were washed twice in PBS-T and incubated overnight at 4 °C with gentle agitation with appropriate anti-species secondary antibodies diluted in immunobuffer (Table S1). Nuclei were counterstained with Hoechst 33258 for 30 minutes and washed in PBS-T. Images were acquired using an automated fluorescent microscope (ImageXpress® Micro XLS; Version 5.3.0.1, Molecular Devices, CA, USA). Quantitative analysis of intensity measures and positively stained cells was performed using the cell scoring and integrated morphometry analysis modules on MetaXpress® software (Version 5.3.0.1, Molecular Devices, CA, USA). Roughly 1000–2000 cells were scored per well with multiple wells (at least three) analysed per sample.

Flow cytometry phagocytosis assay

To investigate the ability of microglia to internalize particles they were incubated for two hours in the presence or absence of a 1:1,000 dilution of 1 μm diameter fluorescent beads (Fluoresbrite® YG Carboxylate microspheres; Polysciences Inc, PA, USA). At completion cells were washed twice with PBS to remove un-internalized beads and collected into a single cell suspension by trypsinisation and gentle scraping as per microglial plating. Samples were run on an Accuri C6 flow-cytometer (BD Biosciences, CA, USA) and viable cells gated based on forward scatter and side scatter. Fluorescent intensity of live cells was indicative of the amount of beads phagocytosed.

Confocal laser scanning microscopy

To confirm internalization of beads for phagocytosis assays, confocal imaging was performed. Microglia were plated on glass coverslips as described above and incubated with a 1:10,000 dilution of 1 μm diameter fluorescent beads (Fluoresbrite® YG Carboxylate microspheres; Polysciences Inc, PA, USA) for 24 hours. Coverslips were immunostained as described under immunocytochemistry and mounted onto glass slides using fluorescent mounting medium (DAKO, Denmark). Confocal images were acquired at a 63 x magnification (1.4 NA) in a Z-series with a gap of 0.8 μm using a Zeiss LSM 710 inverted confocal microscope (Biomedical Imaging Research Unit, University of Auckland).

Cytometric bead array

Conditioned media (70 μL) was collected from cells grown in a 96 well plate. To remove possible floating cells and debris, media was spun at 160 ×g for five minutes and 40 μL was transferred to a new tube and stored at −20 °C. The concentration of secreted cytokine/chemokines (Table S2) was determined using a multiplexed cytometric bead array (CBA; BD Biosciences, CA, USA) run on an Accuri C6 flow cytometer (BD Biosciences, CA, USA) as described previously22,23. Data was analysed using FCAP-array software (Version 3.1; BD Biosciences, CA, USA) to convert fluorescent intensity values into concentrations.

Finnaly, I added the practical article with the method of the isolation of human

microglia.

If you need anythink mre, please contact me!

Sincerely yours!

Dr Bratko Filipič

Email: [email protected]

I would not order primary human microglia from Celprogen, the cells do not look like, or respond like primary human microglia.

Austin Jeffries is right. DONT use the microglia from Celprogen. They are not human microglia. I bought three vials from different donors. All three cell lines did not behavior like microglia. Later on I did RNAseq, which confirmed that >90% of RNAs are CHO genome NOT human. Ridiculously, the company send me a meaningless document to show they are microglia, which makes me doubt that their product scientists never learned biology.

I would avoid Celprogen too, I got a contaminated vial and they refused to replace because we didn't use their plastic/media. Their costumer service is really bad.