Our lab at Kyushu University aim to qualify the laws of microglia dysfunction in various neuropsychiatric disorders. We believe that our IMG cell technology will send neuron on micro dynamic function/dysfunction at the cellular level in human, beyond the limitation of human brain analysis such as postmitotic study and also Pitt brain studies. Even now, animal experiments are essential to qualify the brain pathophysiology of mental disorders.
But it's unclear how well the data can be applied to human pathophysiology. Our human blood in this IMG cell technology will fill this gap between loading data, clinical data, and microglia function and/or dysfunction. The human-derived cellular disease model, human iPS cell drive the microglia cells are also developed, which need much time and much money to develop, and can assess traits markers, but not stem markers.
The advantage of our IMG methods is that it is much simpler, less time-consuming, and cost effective. We have already built several candidate microglia factors by our reverse-translational iMG research focused on neuropsychiatric disorders, such as dementia psychological disease, bipolar disorder, fibromyalgia, gliomas, and many more disease. We believe that our IMG cell technology will send neurons on microdynamic function/dysfunction at the cellular level in various neuropsychiatric disorders.
To begin, put 15 milliliters of the density gradient medium in a 50-milliliter centrifugation tube, and spin it at 1, 000G 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 milliters 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, 000G for 10 minutes at room temperature. Remove the top layer of plasma up to one centimeter above the buffy coat. Decant the remaining the 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 50-milliliter conical tube. Then filter the cell suspension through the cell strainer into the 50-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 250G 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.
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 protend 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 seating 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.
