This work was partially supported by the following Grants-in-Aid for Scientific Research: (1) The Japan Society for the Promotion of Science (KAKENHI; JP18H04042, JP19K21591, JP20H01773, and JP22H00494 to TAK, JP22H03000 to M.O.); (2) The Japan Agency for Medical Research and Development (AMED; JP21wm0425010 to TAK, JP22dk0207065 to M.O.) and (3) The Japan Science and Technology Agency CREST (JPMJCR22N5 to TAK). The funding bodies assumed no roles in the study design, data collection and analysis, decision to publish, or manuscript preparation. We would like to thank Editage (www.editage.jp) for English language editing.

The study protocol was approved by the Ethics Committee of Kyushu University and complied with all the provisions of the Declaration of Helsinki. Written informed consent was obtained from all participants, including healthy volunteers and patients, to analyze their blood and publish their data. Materials and equipment are listed in the Table of Materials, and the compositions of the solutions are detailed in Table 1.
1. Preparation of media and buffers for experiments
<ol>
	<li>Prepare the isolation medium by supplementing RPMI-1640 with 10% heat-inactivated fetal bovine serum (56 &#176;C, 30 min) and 1 % penicillin-streptomycin.</li>
	<li>Prepare the isolation buffer by supplementing the basic buffer solution with 0.5% bovine serum albumin (BSA) stock solution and sterilize it via filtration (0.22 &#181;m).</li>
</ol>
2. Isolation of mononuclear cells from whole blood
<ol>
	<li>Transfer 15 mL of the density gradient medium to a 50-mL density gradient centrifugation tube and centrifuge at 1,000 &#215; g for 1 min at 20 &#176;C. 
	NOTE: Isolate 15 mL of blood per 50-mL density gradient centrifugation tube. Increase the number of tubes when blood volume is high.</li>
	<li>Homogenize the blood collected in heparin tubes by inversion and remove the lid from the blood collection tube with fire-sterilized tweezers.</li>
	<li>Dispense an equal volume (10-15 mL) of blood to the density gradient medium in the density gradient centrifugation tube.</li>
	<li>Set the deceleration level of the centrifuge to 1 of 4 levels and centrifuge at 1,000 &#215; g for 10 min at room temperature (RT). 
	NOTE: At this deceleration rate, a rotational speed of 180 &#215; g (1,000 rpm) arrests in 3 min.</li>
	<li>Prepare an equal number of 50-mL centrifuge tubes as the 50-mL density gradient centrifugation tubes and fill each tube with 10 mL of PBS (&#8722;).</li>
	<li>Remove the top layer of plasma up to approximately 1 cm above the buffy coat following centrifugation. 
	NOTE: Insert an aspirating pipette vertically into the tube and aspirate carefully from the center of the liquid surface. Do not overaspirate to avoid disturbing the buffy coat.</li>
	<li>Decant all of the remaining supernatant from the 50 mL density gradient centrifugation tube into a centrifuge tube containing 10 mL of PBS (&#8722;).</li>
	<li>Reset the deceleration level of the centrifuge to 3 of 4 levels, and centrifuge at 250 &#215; g for 10 min at RT. During this step, prepare one 50-mL and two 15-mL centrifuge tubes and a 100-&#181;m cell strainer.</li>
	<li>After centrifugation, establish the temperature of the centrifuge at 4 &#176;C. Search for a white pellet with a red periphery at the bottom of the centrifuge tube. Remove the supernatant by aspiration and loosen the pellet gently by tapping.</li>
	<li>Position a 100-&#181;m cell strainer on the 50-mL conical tube using fire-sterilized tweezers.</li>
	<li>Incorporate 13 mL of isolation medium into one of the centrifuged tubes and wash the inner wall to collect the cells properly. Take the cell suspension and wash the remaining centrifuged tubes in a consecutive manner. After collecting the cells from all the tubes, filter the cell suspension through a cell strainer into the 50-mL conical tube. 
	NOTE: The isolation medium needs to be chilled throughout the isolation process (steps 2.11-2.16).</li>
	<li>Wash each tube again with 13 mL of isolation medium as described in step 2.11. Collect a total of 26 mL of cell suspension.</li>
	<li>Dispense equal amounts into two 15-mL conical tubes, enumerate the cells, and calculate the appropriate concentration of isolation buffer (60 &#181;L of buffer per 107 total cells) for step 2.16. 
	NOTE: The 15-mL conical tube is better to work with because the liquid volume is small and the liquid bubbles easily when pipetting in step 2.16.</li>
	<li>Centrifuge at 250 &#215; g for 10 min at 4 &#176;C.</li>
	<li>During the centrifugation, thoroughly disinfect the magnetic stand (especially the part in contact with the column) with 70% ethanol and place it on the bench to air dry. Keep the isolation buffer on ice.</li>
	<li>Remove the supernatant by aspiration (at this stage, look for red pellets ~1 mm thick because of the presence of red blood cells), add the appropriate amount of isolation buffer, and loosen the pellet gently by pipetting ~20x.</li>
</ol>
3. Isolation of monocytes using CD11b microbeads
<ol>
	<li>Vortex human FcR blocking reagent for 10 s prior to application. Incorporate the required amount of reagent (20 &#181;L of FcR Blocking Reagent per 107 total cells) and incubate in a chamber at 4 &#176;C for 5 min.</li>
	<li>Vortex the CD11b microbeads for 10 s prior to application. Incorporate the required amount of reagent (20 &#181;L of CD11b microbeads per 107 total cells), and incubate in a 4 &#176;C chamber for 20 min while agitating by hand every 5 min.</li>
	<li>After incubation, add 10 mL of isolation buffer, suspend by gentle pipetting, and centrifuge at 300 &#215; g for 10 min at 4 &#176;C.</li>
	<li>During centrifugation, place the magnetic column on the magnetic stand and rinse it with 3 mL of isolation buffer. 
	NOTE: Refrain from touching the magnetic part of the column and position the column correctly. If bubbles enter the column, remove them with a pipette. The isolation buffer is mandated to be chilled throughout the isolation process (steps 3.4-3.9).</li>
	<li>Remove the post-centrifugation supernatant by aspiration, incorporate 1 mL of isolation buffer, mix by pipetting gently, and apply the suspension onto the column.</li>
	<li>Wash the column 3x by adding 3 mL of isolation buffer each time the column reservoir is empty.</li>
	<li>Place the column in a 15-mL conical tube without touching the magnetic part; incorporate 5 mL of isolation buffer into the column and force the liquid out through the column with a plunger inserted into the tube.</li>
	<li>Centrifuge the cell suspension collected in the tube at 300 &#215; g for 10 min at 4 &#176;C.</li>
	<li>Aspirate the supernatant and resuspend the cells in isolation medium. As ~5-10% of the number of cells counted in step 2.13 will be recovered, adjust the suspension volume according to the number of cells. 
	NOTE: Isolation medium should be used at RT and not warmed to avoid rapid temperature change from 4 &#176;C.</li>
	<li>Enumerate the cells and aliquot 500 &#181;L of cell suspension into each well of 24-well plates at a concentration of 40 &#215; 104 cells/mL and incubate overnight. 
	NOTE: The cell suspensions should be pipetted appropriately in accordance with the adjusted seeding volume required for different plates. For example, 1 mL for one well in a 12-well plate and 250 &#181;L for one well in an 8-well chamber slide.</li>
	<li>Finally, dispose of or disinfect the contaminated equipment in accordance with the policy for handling infectious waste established by the facility.</li>
</ol>
4. Induction of iMG cells from monocytes
<ol>
	<li>Prepare the induction medium by supplementing basal induction medium with 1% antibiotic-antimycotic solution, 10 ng/mL recombinant human GM-CSF, and 100 ng/mL recombinant human IL-34.</li>
	<li>Replace the isolation medium from the seeding of the previous day with induction medium. Tilt the plate forward and promptly aspirate the depleted medium from the front edge of the well bottom with a Pasteur pipette. Replace the medium at the rate of a maximum of 1 plate (= 24 wells) at a time, and immediately incorporate 500 &#181;L of induction medium gently. 
	NOTE: Adhered monocytes should be selected in this step.</li>
	<li>Incubate the cells for 14 days to complete the induction.</li>
	<li>Replace the medium again with 500 &#181;L of induction medium after 14 days. Subsequently, for post-induction maintenance of the cells, replace the induction medium with 500 &#181;L of fresh induction medium every week.</li>
</ol>
5. Immunocytochemistry
<ol>
	<li>Remove the medium from the 8-well chamber slide seeded with cells in step 3.10 by aspiration and rinse with PBS (&#8722;).</li>
	<li>Fix the cells for 20 min at RT with 4% paraformaldehyde.</li>
	<li>Remove the fixative reagent and rinse the cells for 3 x 5 min with PBS (&#8722;).</li>
	<li>Permeabilize the cells with PBS (&#8722;) containing 0.1% Triton X-100 at RT.</li>
	<li>Incubate the permeabilized cells with a blocking solution (3% BSA/PBS (&#8722;)) for 1 h at RT.</li>
	<li>Incubate the cells overnight with primary antibodies (Table of Materials) at 4 &#176;C. 
	NOTE: All antibodies are diluted in blocking solution.</li>
	<li>Rinse the cells for 3 x 5 min with PBS (&#8722;) for 5 min each.</li>
	<li>Incubate the rinsed cells with the appropriate fluorescently tagged secondary antibodies for 1 h at RT (Table of Materials).</li>
	<li>Rinse the cells for 3 x 5 min with PBS (&#8722;) to remove the secondary antibodies.</li>
	<li>Incubate the cells with DAPI (1:10,000) solution diluted with PBS (&#8722;) for 5 min at RT.</li>
	<li>Rinse the cells for 3 x 5 min with PBS (&#8722;).</li>
	<li>Mount the well using the mounting medium and visualize the cells under a fluorescence microscope.</li>
</ol>

Developing Blood-Induced Microglia-Like Cells for Studying Human Brain Disorders

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The authors have nothing to disclose.

Analytical techniques employing iMG cells may serve as potent reverse-translational research tools5,6. To generate sufficient quantities of human iMG cells, experimenters should design their studies taking certain issues into consideration. Blood samples derived from human beings are extremely sensitive; consequently, the obtained samples warrant prompt processing, and meticulous handling to avoid contamination. Specifically, blood samples should be separated imm...

In recent years, brain inflammation has been suggested to assume pivotal roles in the pathophysiology of various brain and neuropsychiatric disorders; the microglia have been highlighted as key immunomodulatory cells by human postmortem brain analysis and positron emission tomography (PET)-based bio-imaging techniques1,2,3,4. Postmortem brain and PET imaging analyses reveal significant findings; nevertheless, however, these approaches are inefficient in terms of capturing the dynamic molecular activities of human microglia in the brain in their entirety. Therefore, novel strategies are required to enable the comprehensive evaluation of human microglial functions and dysfunctions at the molecular and cellular levels.
In 2014, we originally engineered a novel technique to produce directly induced microglia-like(iMG) cells5,6, prior to the first publication of human-induced pluripotent stem cell (iPSC)-derived microglia-like cells in 20167. In just 2 weeks, we successfully converted human peripheral blood monocytes into iMG cells by optimizing the cytokines, granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 34 (IL-34). When we developed this technique, the innovative reprogramming method of inducing neuronal cells from iPS cells or fibroblasts was just beginning to prevail in the world8,9,10,11. However, at that time, a method for inducing iPS-derived microglial cells had not yet been reported, and the generation of a human somatic cell-derived microglial model was desired. Since cytokines such as GM-CSF and IL-34, macrophage colony-stimulating factor, were reported to be necessary for the development and maintenance of microglia12,13,14,15, we hypothesized that a combination of these cytokines could be applied directly to generate a microglial cellular model from blood monocytes. Finally, we succeeded in developing a model of microglia derived from monocytes by combining GM-CSF and IL-345. In addition, some of these combinations of cytokines are also employed to induce microglia from iPS cells7,16 and are assumed to be an important factor in acquiring microglial characteristics.
In contrast to iPSC methods, iMG cells do not require any genetic modification and can be generated in a very short time by simple chemical induction, resulting in lower time and financial costs. Furthermore, iMG cells do not require genetic reprogramming, so we believe that iMG cells are potent surrogate cells to evaluate not only the traits but also the states of human microglia. In the initial paper on the iMG technique in 2014, we confirmed that iMG cells exhibit a phenotype of human microglia, which can be distinct from monocytes and macrophages. For example, iMG cells exhibited an overexpression ratio of CX3C chemokine receptor 1 (CX3CR1) and C-C chemokine receptor type 2 (CCR2) than monocytes and typical microglia markers, including transmembrane protein 119(TMEM 119) and purinergic receptor P2RY125,17. Recently, we validated that peripheral blood-derived iMG cells resemble brain microglia in their gene expression profile of well-known microglial markers in the same patient who underwent brain surgeries18. The iMG cells can be analyzed for dynamic functions at the molecular level, such as phagocytosis and cytokine production, and are expected to compensate for the disadvantages of postmortem brain research and PET studies.
We have discovered previously unknown dynamic pathophysiological mechanisms involving microglia in patients diagnosed with Nasu-Hakola disease5, fibromyalgia19, bipolar disorder20,21, or Moyamoya disease22. Furthermore, based on our original methodology, various laboratories have employed the iMG cells (certain laboratories have designated alternative names to these cells) as a crucial reverse-translational research tool23,24,25,26,27. Sellgren et al. successfully generated iMG cells in compliance with our recommendations and conducted a microarray analysis, which revealed that these cells closely resemble human brain microglia23. Recently, we confirmed the resemblance between human iMG cells and brain primary microglia using RNA sequencing18.
This study aimed to document the methodology to generate iMG cells from human peripheral blood to facilitate reverse-translational research focused on neuropsychiatric diseases. This technique presents potential as a reasonable analytical tool that can effortlessly produce microglial cellular models in a brief duration, even in ill-equipped laboratories that lack gene transfer apparatus or proficient personnel.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.1% Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>30-5140-5</td>
			<td></td>
		</tr>
		<tr>
			<td>4% paraformaldehyde</td>
			<td>Nacalai Tesque</td>
			<td>09154-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic (100x)</td>
			<td>gibco</td>
			<td>15240-062</td>
			<td>described as &#34;antibiotic-antimycotic solution&#34;</td>
		</tr>
		<tr>
			<td>autoMACS Rinsing Solution</td>
			<td>Miltenyi Biotec</td>
			<td>130-091-222</td>
			<td>described as &#34;basic buffer solution&#34; and used for &#34;isolation buffer&#34;</td>
		</tr>
		<tr>
			<td>CD11b MicroBeads</td>
			<td>Miltenyi Biotec</td>
			<td>130&#8211;049-601</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI solution</td>
			<td>DOJINDO</td>
			<td>28718-90-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate Buffered Saline</td>
			<td>Nacalai Tesque</td>
			<td>14249-24</td>
			<td>described as &#34;PBS (&#8722;)&#34;</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>biowest</td>
			<td>S1760-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Histopaque-1077</td>
			<td>Sigma-Aldrich</td>
			<td>10771</td>
			<td>described as &#34;density gradient medium&#34;</td>
		</tr>
		<tr>
			<td>Human FcR Blocking Reagent</td>
			<td>Miltenyi Biotec</td>
			<td>130&#8211;059-901</td>
			<td></td>
		</tr>
		<tr>
			<td>Leucosep</td>
			<td>Greiner Bio-One</td>
			<td>227290</td>
			<td>described as &#34;density gradient centrifugation tube&#34;</td>
		</tr>
		<tr>
			<td>MACS LS columns</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-401</td>
			<td>described as &#34;magnetic column&#34;</td>
		</tr>
		<tr>
			<td>MACS BSA Stock Solution</td>
			<td>Miltenyi Biotec</td>
			<td>130-091-376</td>
			<td>described as &#34;bovine serum albumin (BSA) stock solution&#34;</td>
		</tr>
		<tr>
			<td>MACS MultiStand</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-303</td>
			<td>described as &#34;magnetic stand&#34;</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Nacalai Tesque</td>
			<td>26253&#8211;84</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Gold Antifade Mountant</td>
			<td>Invitrogen</td>
			<td>P10144</td>
			<td>described as &#34;mounting media&#34;</td>
		</tr>
		<tr>
			<td>recombinant human GM-CSF</td>
			<td>R&#38;D Systems</td>
			<td>215-GM</td>
			<td></td>
		</tr>
		<tr>
			<td>recombinant human IL-34</td>
			<td>R&#38;D Systems</td>
			<td>5265-IL</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 Medium + GlutaMAX Supplement (pre-supplemented medium)</td>
			<td>Thermo Fisher Scientific</td>
			<td>61870036</td>
			<td>described as &#34;basal induction medium&#34;</td>
		</tr>
		<tr>
			<td>RPMI-1640</td>
			<td>Nacalai Tesque</td>
			<td>30264-56</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>anti-P2RY12 antibody</td>
			<td>Sigma-Aldrich</td>
			<td>HPA014518</td>
			<td>primary antibody, rabbit, 1:100</td>
		</tr>
		<tr>
			<td>anti-TMEM119 antibody</td>
			<td>Sigma-Aldrich</td>
			<td>HPA051870</td>
			<td>primary antibody, rabbit, 1:100</td>
		</tr>
		<tr>
			<td>Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 568</td>
			<td>invitrogen</td>
			<td>A-11011</td>
			<td>secondary antibody, rabbit, 1:1000</td>
		</tr>
	</tbody>
</table>

engineering of human blood-induced microglia-like cells for reverse-translational brain research

Recent investigations employing animal models have highlighted the significance of microglia as crucial immunological modulators in various neuropsychiatric and physical diseases. Postmortem brain analysis and positron emission tomography imaging are representative research methods that evaluate microglial activation in human patients; the findings have revealed the activation of microglia in the brains of patients presenting with various neuropsychiatric disorders and chronic pain. Nonetheless, the aforementioned technique merely facilitates the assessment of limited aspects of microglial activation. 
In lieu of brain biopsy and the induced pluripotent stem cell technique, we initially devised a technique to generate directly induced microglia-like (iMG) cells from freshly derived human peripheral blood monocytes by supplementing them with granulocyte-macrophage colony-stimulating factor and interleukin 34 for 2 weeks. These iMG cells can be employed to perform dynamic morphological and molecular-level analyses concerning phagocytic capacity and cytokine releases following cellular-level stress stimulation. Recently, comprehensive transcriptome analysis has been used to verify the similarity between human iMG cells and brain primary microglia. 
The patient-derived iMG cells may serve as key surrogate markers for predicting microglial activation in human brains and have aided in the unveiling of previously unknown dynamic pathophysiology of microglia in patients with Nasu-Hakola disease, fibromyalgia, bipolar disorder, and Moyamoya disease. Therefore, the iMG-based technique serves as a valuable reverse-translational tool and provides novel insights into elucidating dynamic the molecular pathophysiology of microglia in a variety of mental and physical diseases.

Author Spotlight: Induced Microglia-Like Cell Technology to Shed Light on the Role of Microglial Dysfunction in Neuropsychiatric Disorders

Engineering of Human Blood-Induced Microglia-like Cells for Reverse-Translational Brain Research

Our lab at Kyushu University aimed to clarify the roles of microglial dysfunction in various neuropsychiatric disorders. We believe that our iMG cell technology will shed new light on microglial dynamic function and dysfunction at the cellular level in human beyond the limitation of human brain analysis, such as postmortem study and also pet brain studies. Even now, animal experiments are essential to clarify the brain pathophysiology of mental disorders, but it is unclear how well rodent data can be applied to human pathophysiology.Our human blood-induced iMG cell technology will this gap between rodent data, clinical data, and microglial function and/or dysfunction. Other human-derived cellular disease model, human iPS cell-derived microglia cells are also developed, which need much time and much money to develop and can assess trait markers but not state markers. The advantage of our iMG method is that it is much simpler, less time-consuming, and cost-effective.We have already revealed several candidate molecular factors by our reverse translational iMG research focusing on neuropsychiatric disorders such as dementia disease, bipolar disorder, fibromyalgia, glioma, and disease. We believe that our iMG cell technology will shed new light on microglial dynamic function and dysfunction at the cellular levels in various neuropsychiatric disorders.

Importantly, there is a great deal of person-level and timing-level heterogeneity in the characters of iMG cells including morphologies and gene expressions. The iMG cells in certain individuals assume a numerous branching appearance (Figure 1A), while in others they remain spherical (Figure 1B). The iMG characteristics may differ even within a single individual, rendering iMG cells as a pivotal tool for detecting disease state biomarkers. Conversely, the examin...

This study delineates a novel approach for the establishment of human monocyte-derived microglia-like (iMG) cells that enable the indirect assessment of brain inflammation. This presents a cellular model that may be beneficial to research focusing on potential inflammation of the brain and associated neuropsychiatric disorders.

Recent investigations employing animal models have highlighted the significance of microglia as crucial immunological modulators in various ...

engineering-human-blood-induced-microglia-like-cells-for-reverse

Research

JoVE Journal

Immunology and Infection

Isolation of Mononuclear Cells from Whole Blood to Obtain Induced Microglia-Like Cells for Brain Research

To begin, put 15 milliliters of the density gradient medium in a 15-milliliter centrifugation tube and spin it at 1, 000 G for one minute at 20 degrees celsius. Invert the tube containing human blood collected in heparin, and using fire sterilized tweezers, remove the lid from the blood collection tube. Dispense around 10 to 15 milliliters of blood to the density-gradient medium in the tube.Set the deceleration level of the centrifuge to one and centrifuge the tube with blood at 1, 000 G for 10 minutes at room temperature. Remove the top layer of plasma up to one centimeter above of the buffy coat. Decant the remaining supernatant from the centrifugation tube into another centrifuge tube containing 10 milliliters of PBS.Reset the deceleration level of the centrifuge to three and centrifuge the tube with PBS and supernatant. Aspirate the supernatant and gently tap the tube to loosen the pellet. Next, incorporate 13 milliliters of isolation medium into the centrifuge tube and wash the inner wall to collect the cells properly.Using fire sterilized tweezers, position a 100-micrometer cell strainer on the 15-milliliter conical tube. Then filter the cell suspension through the cell strainer into the 15-milliliter conical tube. Dispense equal amounts of cell suspension into two 15-milliliter conical tubes.Enumerate the cells to calculate the appropriate concentration of isolation buffer. Pellet the cells at 250 G for 10 minutes at four degrees celsius. After removing the supernatant, add the appropriate amount of isolation buffer and pipette around 20 times to loosen the pellet.

Obtaining Induced Microglia-Like Cells from Whole-Blood Derived Monocytes for Probing Microglial Dynamics in Human Diseases 

To begin, obtain human mononuclear cells from whole blood. Vortex human FcR blocking reagent, and add the required amount of reagent to the cells. Incubate the tube with cells at four degrees Celsius for five minutes.Add 20 microliters of CD11b MicroBeads per 10 to the power of seven total cells, and incubate at four degrees Celsius for 20 minutes. Thoroughly disinfect the magnetic stand with 70%ethanol. After incubation, add 10 milliliters of isolation buffer.Mix, and centrifuge the tube at 300G for 10 minutes at four degrees celsius. Once the stand dries, position the magnetic column on the magnetic stand. After removing the supernatant, add one milliliter of isolation buffer and mix gently with the pipette.Then load the cell suspension onto the column and wash it three times with three milliliters of isolation buffer each time the column reservoir is empty. Place the column in a 15-milliliter conical tube without touching the magnetic part. After adding five milliliters of isolation buffer, push the plunger into the tube to force the liquid out through the column.Centrifuge the cell suspension collected in the tube. Aspirate the supernatant and re-suspend the cells in isolation medium. Following cell counting, aliquot 500 microliters of cell suspension into each well of a 24-well plate, and incubate overnight.To induce microglia-like cells, replace the isolation medium from the seeding of the previous day with an induction medium and incubate the cells for 14 days. The induced microglia-like cells exhibited minute soma bodies and numerous branched collaterals.

712 Views.  Kyushu University.  This study delineates a novel approach for the establishment of human monocyte-derived microglia-like (iMG) cells that enable the indirect assessment of brain inflammation. This presents a cellular model that may be beneficial to research focusing on potential inflammation of the brain and associated neuropsychiatric disorders.

Developing Blood-Induced Microglia-Like Cells for Studying Human Brain Disorders

Summary