TITLE: UABMC - Stemness Determination Protocol
AUTHORS: Christopher Willey

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This procedure describes the assays for determining stem-ness in the PDMC models. </div><div class = "text-block">The phenotypic and marker-based assays that can and will be used for determining/classifying the stem-cell like characteristics of a particular model, namely neurosphere, geltrex (or equivalent) adherent cells, or microtumors.</div><div class = "text-block">Several of these assays are not gold-standard but are semi-validated surrogates for stem-like cells</div></div>

[STEPS]
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Ensure cultures are healthy with minimal percentages of dead cells
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Dissociate cells using Accutase as usual
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While cells are dissociating, add stem cell media to inner wells (6x10) at 200uL per well and to well H2.  Add water to the outer wells at 300uL per well except for well H2.
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Add PI stain to the dissociated cells
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Take to flow cytometry and sort PI- cells into 96 wells
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Plate cells in limiting dilution down to 1 cell per well.
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A good starting template for stem cell enriched cultures is:  AB1B2-B111 cell per well2C2-C115 cells per well3D2-D1110 cells per well4E2-E11 25 cells per well5F2-F1150 cells per well6G2-G11100 cells per well7H21000 cells (+ control to adjust visual plane)A good starting template for bulk cultures is:  AB1B2-B11 1 cell per well2C2-C1120 cells per well3D2-D1150 cells per well4E2-E11 100 cells per well5F2-F11500 cells per well6G2-G111000 cells per well7H25000 cells (+ control to adjust visual plane)
AB1B2-B111 cell per well2C2-C115 cells per well3D2-D1110 cells per well4E2-E11 25 cells per well5F2-F1150 cells per well6G2-G11100 cells per well7H21000 cells (+ control to adjust visual plane)
AB1B2-B11 1 cell per well2C2-C1120 cells per well3D2-D1150 cells per well4E2-E11 100 cells per well5F2-F11500 cells per well6G2-G111000 cells per well7H25000 cells (+ control to adjust visual plane)
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For repetitions, adjust as needed based on prior results by increasing the number of wells in the ranges where the sphere formation is less than 100% but greater.  Once you have identified working conditions, increase the # of wells to be at least two rows (20 wells) of each to gain statistical power. than 0%. Keep numbers so that one row is expected to have 100% spheres.
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Filter through a 0.45 micron filter and then plate this media into the 96 wells with 100uL conditioned media and 100uL fresh media per well. If flow cytometry is a problem due to either inaccurate cell numbers (check immediately post flow), viability post flow (check 24hrs post flow) or contamination, you can plate the assay by hand.  In this case, if you are comparing two cell types (rather than completing a drug treatment) you must count the cells in at least triplicate prior to initiating the assay to ensure correct counts.  Dilute cells to 5000 cells/200uL= 250000 cells/10 mL and plate well H2.  Then dilute from this stock in a stepwise fashion as needed: ie 5000 cells/200uL to 1000 cells/200uL.  Then 1000 cells/200uL to 500 cells/200uL etc.
If the above method has not worked previously, save the conditioned media from the cells prior to dissociation.
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Check plates at 7, 14, and 21 days.  Note the number of wells with spheres then use this information along with the total number of wells and cells plated to complete Statistical Analysis at http://bioinf.wehi.edu.au/software/elda/
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At an optimal time point for one cell number condition (need condition where spheres are individual and no merging has occurred, for example 2nd lowest cell number where spheres formed at 14 days), can also collect data for the number of spheres per well as well as the size of the spheres formed.   However, these data are not as important for publication as the limiting dilution analysis.
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If Miltenyi tumor dissociation kits are used, cells can be sorted immediately.  If Papain is used for tumor dissociation, ensure that cells have recovered at least  but preferably .  If cells in culture are dissociated with Accutase, ensure that cells have recovered at least 6 hrs but preferably overnight.  Regardless of the tissue dissociation protocol, ensure cells are healthy prior to beginning.
For analysis only. If sorting for propagation, MACS rather than FACS is recommended.
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Ensure cells are single cells for flow.  If flow is not performed shortly after dissociation, triturate cells by passing through a P200 tip at least 20 times and then pass through a 40 um filter.
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Wash cells in naught media, count cells and resuspend in  naught media and  FcR Blocking Reagent for every 10 million cells.
400 µl
400 µl
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Distribute cells to appropriate tubes for no staining, viability dye alone, isotype control along, and antibody and viability labeling. For the first three tubes, only a very small portion of cells are required (at least 100,000 cells).
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When using the antibodies, ensure the hood light is off and tubes are covered in aluminum foil/kept in the dark.
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Add the antibodies or appropriate controls.  As Milteny isotype antibodies are  , use  of these isotype controls.  Add  of CD133/2 (293C3)-APC antibody for up to 10 million cells. Mix by shaking the tube and then incubate for  at , mixing by shaking the tube every 5 min.
1 µl
50 µl
0 Room temperature
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After the , wash the cells at  with of naught media for
Centrifuge: 1000 33
3 mL
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Resuspend the cells in 5% BSA with pen/strep. Use for 10 million cells or  for controls.
500 µl
200 µl