Neural differentiation of human EC and ES cells
Neural differentiation of human EC and ES cells
The study of both E S and E C cell differentiation has facilitated the development of experimental techniques. E S cells are very sensitive to differentiation induction and cell death, making the establishment of experimental methods a long and arduous process. The distinctive features of E C cells and their undifferentiated nature similar to E S cells make them ideal cell lines for establishing experimental techniques prior to E S experiments. We have found this to be particularly true for the establishment of techniques such as RNAi. Author: Stacey et al., Translated by Jingbo Zhang, from "Human Stem Cell Culturing
Operation method
Neural Differentiation Techniques for Human EC and ES Cells Move I. Preparation of bases and reagents 1. Culture medium IDMEM /10F B and D M E M /20FB DMEM/10FB , DMEM, 4. 5 g/L glucose with 10 % fetal bovine serum. DMEM/20FB: DMEM, 4.5 g/L glucose, containing 20 % fetal bovine serum. Human embryonic stem cell (h ES) medium (approx. 500 m l) Knockout DMEM 400 ml Knockout Serum Substitute 100 ml FGF-2 4ng/ml Non-essential amino acids, IOOX 5 ml β-mercaptoethanol 3.5ul L-glutamine I .Ommol/L Neurosphere proliferation medium (100m l ) DMEM/F12 99 ml N2 supplement I ml FGF-2 20 ng/ml Insulin 20 ug / m l Heparin sodium sulfate 2ug/ml Neurosphere culture base (100m l ) DMEM/F 12 97 ml B2 7 Supplement 2 ml Insulin-Transferrin-Smash Supplement Iml FGF-2 20ng/ml E C Cell Differentiation Basis DMEM/F12 (see Section 3.4.1.1) supplemented with 10 mmol/L all-trans retinoic acid (ATRA). All-trans retinoic acid is photosensitive. It should be handled under dim light. Dissolve in DMSO, store at -20°C in a 10 mmol/L storage solution and dilute 1:1000 before use. EB medium (approx. 500 m l ) Knockout DMEM Knockout Serum Replacement Non-Essential Amino Acids, 100X β-Mercaptoethanol L-Glutamine Collagenase Collagenase IV, 10 mg, dissolved in 10 ml of DME/F 12, final concentration l m g/m l, filtered for sterilization. Neutral protease Dilute the neutral protease stock solution 1:4000 with Ca2+ and Mg2+ free phosphate buffer (PBSA). Trypsin/E D T A Trypsin, 0.05% (W /V), ED TA, 0.5 mmol/L, dissolved in PBSA. Agar Sugar Agarose, 1% (W /V), soluble in D M E M /F 12 (see Section 3.4.1.1). Gelatin layer Gelatin, 0. 1 % (W /V ) , soluble in P B S A . Add the gelatine solution to the culture flasks or petri dishes, let stand for 5-10 min, remove, and allow the flasks/m to dry. Flow Cytometer Flush Buffer (a) Fetal bovine serum, U.S. origin, prepared to 5 % with PBSA. (b) Fetal bovine serum, U.S. origin, prepared to 5% with PBSA and 0.1% (VVV) TritonX-100. (c) Poly(cockroach aldehyde), 4% (W/v) 4 g paraformaldehyde dissolved in iooml PBSA, stirred and heated until completely dissolved; stored at 4 °C. Triton solution Prepare a 0.1% ( V A O T r i t o n X -1 0 0 and 5% fetal bovine serum with PBSA. Triton X-100 buffer with goat's serum P B S A contains 1 % (v/v) Triton X-100, l m o l / L Glycine, 5 % normal goat serum. Blocking solution PBSA containing 5 % FBS, 0.1 % (V/V) Trioton X-100. N T E R A 2 Cells Freezing and Resuscitation Reagents and Materials Sterilization □ DMEM/10FB (see Section 3. 4. I.1) □ Freeze solution: 90 % FB S, 10 % DMSO (V/V) Glass beads, 3 mm Steps (a) Mechanically disperse the cell culture with glass beads and resuspend the cells with DMEM/10F B (see Scheme 3.1). (b) Place the suspended cells in a sterilized tube and centrifuge at 12000 r/min (277 g) for 3 min at 4°C. (c) Aspirate the culture medium and resuspend the cell pellet with freezing solution. (d) Aliquot the cell suspension into Im l freezing tubes. (e) Place the frozen tubes into a Nalgene freezer box and place at 8(TC) for at least 24 h. The frozen tubes are then placed into a Nalgene freezer box. (f) When the tubes reach -80 °C, transfer to liquid nitrogen storage. (g) Remove the cryopreserved material from liquid nitrogen and thaw rapidly at 37°C. (h) Cells were washed with 10 ml DMEM/10FB to remove residual DMSO, 1200 r/min (277 g), centrifuged for 3 min. (i) Resuspend cells with 5 ml of fresh culture medium and inoculate into 25 cm2 flasks. Freezing and recovery of human ES cells Confluent cells were harvested from 25 cm2 culture flasks in the usual way (see Chapter 2). The cells were transferred to 15 ml tubes with IOml culture medium, and the procedure for freezing NTERA2 cells was followed, except that the centrifugation conditions were changed to 800r/min (123 g) for 3 min at 4°C. The thawing temperature was the same. For more information, see Chapter 2 on freezing and thawing of human ES cells. ES cells Retinoic acid-induced differentiation of human E c cells Reagents and Materials Sterilization or sterile preparation □ NTERA2 cell confluent cultures □ DMEM/IOFB □ A T R A , 10mmol/L dissolved in DMSO; -20°C storage □ Differentiation medium: DMEM/10F B, with 10 mmol/L ATRA (see section 3. 4. 1. 5) Trypsin/EDTA (see section 3. 4. 2. 3) □ Culture bottle, 75c m 2 Steps (a) Incubate at 37 °C with 2 ml of trypsin-EDTA digest until the cells are dispersed from the bottom of the culture flask. (b) Inactivate trypsin activity by adding 8 ml of DMEM/10FB. (c) Centrifuge at 1200 r/min (277 g) for 3 m i n . (d ) Inoculate cells I X l o 6 per 75 c m 2 culture flask with differentiation medium. (e) Change the differentiation medium every 7 days. Cells begin to differentiate within 2 to 3 days, and after 7 days the cells show very specific morphological changes and surface antigenic phenotypes; neurons begin to appear within the second week. ES cells Differentiation of human ES cells in embryonic bodies (EBs) Reagents and Materials Sterilization □ DMEM/10FB □ Collagenase IV solution, l m g / m l (see section 3. 4. 2. 1) □ Glass beads, 3 m m □ Petri dish, bacteriological grade, IO cm (78.5 cm2) □ Centrifuge tube, 15 ml □ gelatinized culture jars or dishes (see section 3.4.3.2) Steps (a) Confluent growth of human E S cells in 25 cmz tissue culture flasks. (b) Aspirate the culture medium from the flask and add I ml of collagenase to ensure that the collagenase covers all cell growth surfaces, then transfer to a 37°C, 5 % CO2) 2 oven for 15 min. (C) Remove the flask from the incubator, check to make sure that the edges of the cell colonies are curled, and add approximately 20 glass beads to the flask. (d) Gently shake the culture flask from side to side to dislodge the cells from the walls. (e) Add 9 ml of DMEM/10FB and resuspend the cells by grinding 5 to 10 times. (f) Pipette the cell suspension from the culture flask into a 15 ml sterile tube. (g) Centrifuge at 800r/min (123 slices) at 4°C for 3million. (h ) IOml E B culture solution to resuspend the cell clumps and transfer the suspension into a 3. 5c m bacterial Petri dish. This step is necessary; the use of a bacterial culture dish prevents the E B cells from adhering to the bottom of the flat dish. (i) Place the dish in a 37°C, 5 % CO2 oven. (j) Continue to culture the suspended cells, replacing the EB culture medium with fresh EB every 2 days. i) Transfer the cells and culture medium to a 15 ml tube. ii) Allow E B s to settle naturally for l0 m in . iii) Pipette off the upper layer of culture medium and resuspend the cells with I O m l of fresh culture medium. iv) The E B s were then transferred to a flat dish. (k) After 21 days of suspension culture, the EBSs should be fully differentiated by this time. Spontaneous differentiation can be seen before 21 days, but human EBs cells may be present earlier. Gelatin is an effective substrate for adherence and growth of initially differentiated cells of EBs. i) Remove the EBs from the dish as if replacing the culture medium. ii) Re-inoculate the E B s cells with E B culture medium into gelatin-covered culture flasks/dishes. The inoculation density depends on the size of the EBS (different normal culture ranges), but usually about 50 EBS per 25 cm2 is sufficient. iii) Spontaneous differentiation is seen in the growth of cells that are reattached after EBS inoculation. Neurospheres Option 3.5 Inducing neurospheres from human ES cells Reagents and materials Sterilization □ DMEM/10FB (see section 3. 4. I.1) □ EB culture fluid (see section 3. 4. 1. 6) □ Neurosphere expansion culture fluid (see section 3.4.1.3) □ Collagenase W solution (see section 3.4.2.1) □ Neutral protease, 0.lmg/ml (see section 3.4.2.2) □ Glass beads, 3 mm □ culture flask, 25 cm2 □ culture flask, 25 cm2, gelatin-coated (see section 3. 4. 3. 2) □ Centrifuge tube, 15 ml (C) Place the flask in a 37°C, 5 % CO2 oven for 15 min. (d) Remove the flasks from the incubator and check to make sure that the edges of the cell colonies are curled. (e) Add approximately 20 sterilized glass beads to the bottle and gently shake from side to side to dislodge the cells from the bottom surface of the bottle. (f) Add 9 ml of DMEM/10F B culture medium to the bottle and resuspend the cells by shaking 5 to 10 times repeatedly. (g) Pipette the cell suspension from the bottle and add it to a 15 ml sterilized tube and centrifuge at 4°C, 800r/min (123 g) for 3niin. (h) Aspirate the supernatant, add 5 ml of EB culture medium and resuspend the cell pellet. (i) Transfer the suspension to gelatin-free 25 cm2 culture flasks and incubate at 37°C in a 5 % CO2 oven for 4 days. (j) The culture medium was changed daily by transferring the floating cell pellet and culture medium to a 15 ml sterilized tube. (k) Allow the cell pellet (EBS) to settle naturally for 15 min. (l) Aspirate off the old culture medium and add fresh culture medium. (m) Re-transfer the cell suspension to a cell culture flask and place in a warm box. Phase 2 (4 days) (a) After 4 days of suspension culture, the embryo samples were transferred to 15 ml tubes and allowed to settle for 15 min. (b) Aspirate off the old culture medium and add 5 ml of neurosphere amplification medium. (c) Inoculate each gelatin-covered 25 cm2 culture flask with approximately 50 EBs and bring the total volume of neurosphere expansion medium to l0 ml per flask. (d) Incubate the flasks at 37°C in a 5 % CO2 incubator for 4 days. (e) Change the culture solution every 2 days by aspirating the old culture solution and adding IOml of fresh neurosphere amplification medium. Phase 3 (U days) (a) After 10 days of phase 2, the neuronal rosettes appearing in each vial were examined under a phase contrast microscope (Fig. 3.1: ). Agarose-coated (a) Agarose encapsulation is a cost-effective way to prevent unwanted adherence of suspension cultures in cell culture flasks (compare to other methods). During cell incubation, 1 % (W/V) agarose is prepared with DMEM/F 12 and heated in a microwave oven at maximum power for 30-40s until dissolved. (b ) Cool at room temperature for no more than IOrnin and then coat the bottom surface of the cell culture flask with agarose. It is important that the agarose is hot enough to not begin to solidify; if it is too hot, it causes the plastic of the culture flask to curl. (c) Allow to stand at room temperature for 20-30m i n . (d ) Add 5m i of neurosphere culture solution containing neuronal rose bengal knots to each agarose-coated culture flask. (e) Place in a 37°C, 5 % CO2 incubator. Maintenance of neurosphere cultures Reagents and Materials Sterilization neurosphere amplification culture solution (see section 3.4.1.3) □ 25 cm2, agarose-coated culture flasks (see Option 3.5, Agarose-coated) □ centrifuge tube, 15 ml Steps Breeding (a) Replace fresh neurosphere culture solution every 3 to 5 days (depending on the size of the neurosphere): i) Transfer the suspension of neurospheres into a 15 ml tube and allow the spheres to settle naturally for 10-20 min. ii) Aspirate off the old culture medium and add IOml of fresh neurosphere culture medium. iii) Transfer the neurosphere culture back to the agarose-coated flask and return the flask to 37°C, 5 % CO2 incubator. (b) For too long, the neurospheres will gather into a large clump, making inoculation and passaging difficult. Therefore, it is essential to thoroughly disperse any cell clumps on a stirring table when culturing or passaging. Passaging of neurosphere cultures Passaging of neurosphere cultures is performed every 2 to 3 weeks, or when they are of a certain size. It is important not to have too many spheres in the same culture flask, as they tend to aggregate when cultured for too long. i) Remove the spheres from the culture flasks, allow them to settle naturally, and replace the old culture medium with 10 ml of fresh neurosphere culture medium as in regular culture. ii) Prepare 2 new agarose-covered culture flasks for each culture flask to be passaged. iii) Transfer neurospheres from one centrifuge tube to 2 25 cm2 culture flasks with fresh culture solution and replenish the culture solution to a total volume of 10 ml. iv) Incubate at 37°C in a 5 % CO2 incubator. Reinoculation of neurosphere differentiation Reagents and Materials Sterilization □ Neurosphere cultures or agarose-coated culture flasks □ neurosphere culture solution (see section 3. 4. 1. 4) □ Trypsin, 0.05% (W/V), 0.5 m m o l / L E D T A □ gelatin-coated 25 cm2 culture flask (see section 3. 4. 3. 2) Steps Reinoculate (replating) (a) Depending on the size of the neurospheres and the size of the container to be inoculated, remove the required number of neurospheres from an agarose-coated 25 cm2 culture jar. For example, 20 neurospheres with a diameter of approximately I m m fit into a 25 cm2 culture jar. (b) Gelatin-coated 25 cm2 culture flasks are inoculated with neurospheres and allowed to attach and grow for 3 to 4 days, replacing the neurosphere culture medium with fresh neurosphere culture solution every other day. At this point, when individual cells are dispersed from the spheres, settle down and begin to proliferate, clear neuronal differentiation is observed and a series of morphological changes are exhibited (Fig. 3 . 2; Color Fig. 1). 2 ; Color Figure 1). Reinoculated neurosphere passages (a) After 3 to 4 days, when the neural protrusions have overgrown and covered the bottom area of the flask, aspirate the culture and add }1111 trypsin/ED T A for 5 min. (b) After 5 min, most of the cells can be seen floating in the supernatant. Add gmiDMEM/lOFB and shake the supernatant several times to dislodge the cells from the bottom of the bottle and further disperse the cell clumps. (c) Transfer the suspension to a 15 ml tube and centrifuge at 4°C, 800r/min (123 g) for 3mn. (d) Remove the supernatant and resuspend the cell pellet with 9 ml of neurosphere culture medium. (e) Add 3 ml of cell suspension to 3 gelatin-coated culture flasks (1 : 3) and top up each flask to a total volume of 10 ml. (f) Incubate the flasks at 37°C in a 5 % CO2 incubator. (g) After about 3 generations, residues of neurospheres will still be present in the culture flasks. This can be gradually eliminated by successive passages until monolayer cells are formed. Maintenance of Reinoculated Neurospheres (N S Cells) After several passages, cells of neurosphere (NS) origin show an advantage in culture. Although they cannot be recognized as neurons based on their morphology, they have the morphological hallmarks of early neuronal starvation, such as musashi-1 and nestin (see Figure 3.2). (a) Every second day, aspirate the old culture medium and add 10 ml of fresh culture medium. (b) Every 3 to 4 days, digest with 0.05% trypsin and 0.5 mmol/L EDTA. (b) Every 3 to 4 days, digest the passages with 0.05% trypsin and 0.5 mmol/L EDTA for 5 min, which is the same as the method of neurosphere re-inoculation. For NS cells, a 1:3 ratio of vials was sufficient. Intracellular antigen Antibody coloring of intracellular antigens Reagents and Materials Non-sterile □ P B S A Polyformaldehyde (see section 3.4.4.2) □ Blocker: (V/V) Triton X-100 0.1% (V/V) prepared with sheep serum and PBSA. Primary antibody in blocking agent □ secondary antibody in blocking agent Steps (a) Wash the cells gently with P B S A . (b) Mix with 4 % paraformaldehyde and leave for 20 min at room temperature. (c) Wash the cells again with PB SA and incubate with blocking agent for 10 min. (d) The blocking agent is aspirated and the blocking agent containing the relevant primary antibody is added, the appropriate dilution of which is determined by titration. (e) Incubate at room temperature for I h or overnight. (f) After primary antibody incubation, wash cells 3 times with PBSA. (g) Add blocking agent containing the relevant secondary antibody and incubate for 1h at room temperature. (h) Nuclei were stained with Hoechst33342 (10 Mg/ml) for 5 min. (i) P B S A washed and stored in P B S A for observation. Bromodeoxyuridine (B r d U ) Staining An important aspect of characterizing and detecting cell differentiation pathways is to identify specific cell types. To determine whether a cell belongs to the post mitotic phase or remains proliferative, by doping with a human thymidine deoxyriboside analog (BrdU) , it is possible to accurately evaluate whether the cell is undergoing proliferation, i.e., whether the cell continues to undergo DNA replication. Using Option 3.9, BrdU can be evaluated to determine whether a cell is proliferating or not. 9, BrdU doping can be evaluated by re-doping and fluorescence staining of protein expression (Fig. 3.3; Color Fig. 2). The length of BrdU's duration of action limits how the results can be interpreted: a short action of BrdU for lh enables the proliferation of cells with a specific phenotype (labeling index), whereas a longer action (24 h) allows the estimation of the number of actively dividing cells (growth fraction). Option 3. 9 Determination of labeling index and growth fraction of differentiated neuronal cells Reagents and materials Sterilization □ Neurospheres or re-inoculated neurosphere growth cultures □ PBSA □ BrdU, l0 mmol/L, dissolved in appropriate growth medium Non-sterile HC1, lmol/L and 2mol/L I5B S A Triton X-100: P B S A contains 0.1 % (V/V) Triton X-100. □ Borate buffer, 0.l m o l / L □ PBSA containing Triton X-100, glycine and normal goat serum (see section 3. 4. 4. 4) □ anti-BrdU antibody □ Hoechst33342, lOug/ml, PBS preparation Steps (a) Remove the growth medium and add culture medium containing B r d U . (b) Incubate for the required time (lh for labeling index experiments and 24 h for growth fraction experiments). (c) Cells were washed with PBSA. (d) 4% paraformaldehyde fixed cells at 4°C for 30 min. (e) Cells were washed three times with PBSA containing 1 % Triton X-100 for 5 min each time. (f) Cells were incubated with lmol/L HC1 on ice for lOmiti to open the DNA structure of labeled cells. (g) 2mol/LHCl for 10 min at room temperature. (h) Incubate at 37°C for 20 min. (i) Add borate buffer to neutralize the acid. (j) Incubate cells at room temperature for 12 min. (k) Wash the samples with PBSA/Triton X-100 3 times for 5 min each at room temperature. (l) Incubate with PBSA containing TritonX-100, glycine and goat serum for 30 min. (m) At 4°C , cells were incubated overnight (Nestin or T U Jl) with antibodies against -B rd U or compounds of anti-B rdU and other antibodies. (n) P B SA/Triton X-100 cells were washed 3 times for 5 min each time. (o) Cell specimens can be used in a series of secondary antibodies to reveal anti-BrdU labeled cells. (p) Incubate cells with PBSA containing Hoechst33342 10/xg/ml for 5 min. (q) PBSA washed and stored the cells for analysis by flow cytometry. Flow cytometry Cell surface antigen Unfixed single cell suspensions stained by indirect immunofluorescence can be analyzed by flow cytometry. Option 3.10 Flow cytometry analysis of neuronal cell surface antigens Reagents and Materials Sterilization Trypsin/EDTA (see section 3. 4. 2. 3) Non-sterile □ -anti, secondary antibody □ flow cytometry wash buffer (see 3. 4. 4. Ia) □ FA CS Buffer □ FACS tubes Steps (a) Im l trypsin/ED TA act on the cells for about 2 min to obtain a single cell suspension. (b) Add 9 ml of Wash Buffer to the cell suspension and shake gently until all clumps are dispersed into single cells. (c) A blood counting plate is used to determine the cell concentration. (d) Centrifuge at 4°C, 1200r/min (277 g) for 3 min. (e) Aspirate the supernatant and resuspend the cells with flow cytometry wash buffer and bring the cell concentration to 5X 106 cells/ml. (f) For 96-well plates, add 50M 1 cell suspension per well. (g) The antibody is diluted to the appropriate volume of working solution (diluted with flow cytometry wash solution) and 50ul is added to the wells containing the cells.As a negative control, either no primary antibody is added or the antibody is replaced with a non-specific antibody produced by the parental myeloma cell line P3X63Ag8. Perhaps the use of a comparable antibody that does not bind to the cells may be considered necessary by some. (h) Seal the plate with a sealer and place it on a shaker at 4°C for lh. (i) After the initial incubation, centrifuge the plates for 3 min at 4°C, 1200r/min (277 g). (j) Remove the sealing film and aspirate the antibody-containing solution, leaving the cell mass behind. (k) Wash the cells 3 times with 100ul of washing solution. (l) Dilute the secondary antibody and add 50ul to each well, then return the plate to the shaker and shake at 4℃ for lh. (m) After incubation, centrifuge and wash the cells 3 times, then resuspend the cells in FA CS tubes with 5OOO FA CS buffer. (n) Flow cytometry analysis. Non-individual cells, cellular debris in front-scattered light, and dead cells in backscattered light were omitted. At least 1000 cells were analyzed per sample. Intracellular Antigen Except for the requirements of cell fixation and permeabilization, the detection of intracellular antigens such as S 〇 x2 by flow cytometry is very similar to the analysis of cell surface antigens. Option 3. 1 1 Analysis of intracellular antigens in neuronal cells by flow cytometry Reagents and materials Sterilization Trypsin/EDTA (see section 3. 4. 2. 3) □ PBSA Non-sterile Polyformaldehyde, 4% (W/V) (see Section 3.4.4.2) □ Blocking Fluid (see Section 3.4.4.5) □ Flow cytometry detergent (see section 3.4.4.Ib) Procedure (a) Trypsin digest the cells, resuspend, and homogenize with 4% paraformaldehyde for 20 min at room temperature. (b) The cells were washed twice with PBSA, centrifuged at 1200 r/min (277 g) for 3 m i n , and collected. (C) Resuspend the cells with blocking solution to a concentration of 5X 105 cells/m l and incubate for lOmin at room temperature. (d) Add 50 mi of the appropriate dilution of antibody to 50 ul of cell suspension to give a total volume of suspension. (e) At 4C, allow to stand for lh. (f) Cells were washed and stained with secondary antibody exactly as in (i) to (n) in Option 3.10. Remove the Triton X-IOO (0.1 %, VAO) contained in the secondary antibody staining solution. 1 %, VAO) in the secondary antibody staining solution (see section 3.4.4.1b). (g) Stained cells are visible under a fluorescence microscope (Fig. 3.4; Color Figure 3). 4; Color Figure 3). Classification of fluorescence-stimulated cells Fluorescence-activated cell sorting (FACS) can be used to obtain purified cell types based on cell surface antigen expression (NCAM, A2B5, etc.). We found that FACS is valuable for the purification of undifferentiated cells prior to differentiation, as well as for the purification of differentiated cell populations. Once live sorted cells are obtained, they can then be analyzed by PCR or Western hybridization, or maintained alive for further analysis. Option 3.12 Incineration-activated sorting of neuronal cells Reagents and materials Sterilization □ Trypsin / E D T A (see section 3. 4. 2. 3) □ Primary and secondary antibodies □ D M E M /10F B (see section 3. 4. 2. 3) □ D M E M /20F B : D M E M , 20 % F B S □ Gentamicin, 50 mg/ml (IF: stock solution) □ F AC S Buffer (P B S A with 10% F B S ) □ FACS tube, 5 ml, 75X 12 m m Steps (a) Trypsin digestion of cells into single cell suspension. (b) Excluding antibodies diluted with normal growth medium and washed cells, cells are stained with primary and secondary antibodies as described in the flow cytometry assay (see Scheme 3. 1 0 ), proportionally increasing the number of cells and volume of antibodies. (c) After antibody staining, cells are analyzed and sorted using a suitable flow cytometric photometer. (d) Sorted cells are collected in supplemental culture medium containing 20% fetal bovine serum. Once the cells have been collected and are to be used for molecular analysis or when a burst of light is seen under a burst light microscope, the cells are inoculated into normal growth medium containing antibody (gentamicin 50ug/ml) (Fig. 3.5; Colour Figure 4). 5; Color Figure 4). During the FACS period, the culture medium was used according to the cell type; NTERA2 cells were treated in serum-containing medium, whereas human ES cells were treated in their normal growth medium. Neuronal Counting E C and E S cells show a typical differentiated state when grown at high density, which makes it difficult to count the number of neurons by the naked eye. Thus, in order to easily count neurons, we inoculated the cells at a lower density before performing immunofluorescence staining. Option 3. 1 3 Counting cultured neurons Reagents and materials Sterilization □ D M E M /10F B □ Trypsin/EDTA (see section 3. 4. 2. 3) □ T U J l antibody □ Multi-well culture plate, 1 2 wells (3c m 2/well) Non-sterile Paraformaldehyde, 4 % (W/V) □ Hoechst 33342 DNA dye, 50n g / m l Steps (a) After NTERA2 cells have been induced by retinoic acid (see Scheme 3.3) for 21 days, trypsinize and resuspend the cells in DMEM/10F B. The cells are then digested with trypsin. (b) Cells were inoculated into 12-well plates at a density of I X l O5 cells/cm2 and incubated for 48 h to allow for cell attachment and easy axon generation. (C) Cells were fixed with 4 % paraformaldehyde for 20 min at room temperature. (d) T U Il staining (see Scheme 3.11). (e) TUJl - once stained, restain with Hoechst 33342 and count the number of nuclei as the total number of cells. (f) Neurons were identified by their reactivity to T U J l . Neurons were identified on the basis of their morphological characteristics (small cytosol, at least one protrusion). For total cell counts and neuron counts, select at least 5 individual fields of view with more than 500 cells. The ease of culturing NTERA2 cells and the reliability of producing a predictable percentage of neurons makes this cell line ideal for studying cell differentiation in a high-throughput system. Pharmaceutical companies have long utilized high-throughput technology to screen compound libraries to identify potential drugs. It is now becoming increasingly appropriate for basic laboratories to use these automated techniques [Abraham et al. 2004; Fennell et al. 2006]. The advantage of automated systems and the use of 96-well plates is the ease of exploring a wide range of cell culture parameters (substrate adhesion, type of base medium, additional media components, growth factors, etc.). Processes such as neuronal differentiation and cell analysis can be accomplished through the use of f For more product details, please visit Aladdin Scientific website.
III. Freezing 1.NTERA2
□ E B culture medium: human ES cell culture medium without F G F-2 (see section 3. 4. 1. 6)
(as per Zhang et al. [2001])
