Education

Differentiation of PC12 Cells

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Science's STKE  05 Sep 2006:
Vol. 2006, Issue 351, pp. tr9
DOI: 10.1126/stke.3512006tr9

Abstract

This 3-week-long series of collaborative laboratory exercises explores how to use a cultured cell system (PC12 cells) to study signaling pathways involved in cellular differentiation. The laboratory would be useful in a neurobiology or cell biology course for advanced undergraduate students. The background and details for performing the lab are provided along with suggestions for assessing student performance and understanding.

Information for Instructors

Overview

This series of laboratory exercises allows undergraduate students in an advanced neurobiology, neuroscience, or cell biology course to use a cultured cell system to investigate how different signaling pathways contribute to cellular differentiation. The exercises are designed for a lab course in which there are two sections, one that meets on Tuesday and one that meets on Thursday. It is best if each section has no more than nine students. The experiments take 3 weeks to complete. Each lab section is divided into three groups, with each group treating cells with different combinations of growth factors and pharmacological agents. The "Introduction" section provides background information that introduces the students to cultured cells in general, and PC12 cells in particular, and describes cell culture technique.

The first week is a demonstration laboratory in which the instructor shows the students how to passage cells and count them using a hemocytometer. Change Bioscience has a hemocytomer calculator with a training option that may be helpful (http://www.changbioscience.com/cell/hemo.html). The students then practice counting the cells and sterile technique.

The second week, the students seed the cells into 6-well plates and work collaboratively to replace the medium and record any changes in morphology. Depending on the number of students in the lab and access to cell culture hoods, all groups may not be able to work simultaneously. In this case, different groups can be scheduled to arrive in the laboratory at different times. The instructor maintains the PC12 cells in culture and provides each group with a 100-mm dish of cells and the collagen-coated 6-well plates.

The third week, the students work collaboratively to replace the medium and record any changes in morphology. The cells are harvested and stored for use in a later laboratory to measure acetylcholinesterase (AChE) activity or transcript abundance by polymerase chain reaction.

Details for each laboratory session are provided, along with what data each group should collect for their experimental samples. Differentiation can be monitored by recording morphological changes (described here), by assaying for the activity of AChE, or by assaying for changes in the expression of AChE transcripts.

Back-Up Cultures, Supplies, and Equipment

If the laboratory sessions are scheduled for Tuesdays and Thursdays, the instructor will have to replace the medium with fresh medium (containing the appropriate growth factors and pharmacological agents) over the weekend. In addition, because students generally have limited experience with sterile technique, it is recommended that the instructor maintain a back-up set of cultures in case contamination becomes a problem for any or all of the student groups.

Cells are handled using sterile technique, which requires a laminar flow or other sterile culture hood and 70% ethanol for decontamination of surfaces. Cells are grown in 6-well plates in a 37°C incubator at 5% CO2. The base medium is Dulbecco’s modified Eagle’s Medium containing 10% fetal calf serum and 5% heat-inactivated horse serum to which the various agents are added. The pharmacological agents described here include nerve growth factor (NGF), pituitary adenylate cyclase-activating polypeptide (PACAP), dexamethasone, forskolin, Rp-cAMPs, and U0126; however, any agents that influence differentiation or promote or interfere with the signaling pathways that are involved in PC12 cell differentiation can be used. The laboratory can be readily modified to investigate other factors that influence cell morphology, such as culture substratum or a signaling pathway of particular interest, depending on the topics covered in lecture.

PC12 cells may also be grown in base media other than Dulbecco’s modified Eagle’s Medium, and serum concentration may be reduced during the period of experimental analysis. Various sublines of PC12 cells are available. Well in advance of the student laboratory, the instructor should determine that the particular subline in hand responds to the various agents under investigation, grows well under the particular culture conditions used, and adheres sufficiently well to the substratum to survive occasional rough handling in a student lab. Some sublines are sufficiently adherent as to require trypsinization for subculturing, which would require modification of the plating instructions to include this step, along with a centrifugation step to remove the trypsin..

Cell morphology may be assessed using compound microscopes in phase contrast mode. Neurite length may be conveniently measured in a system using a video monitor with a flexible wire held up to the monitor, with actual length determined by comparison to a stage micrometer. Cell number is assessed by counting the cells using a hemocytometer.

The instructor may prepare the sterile solutions (basic medium and the medium with the agents) and aliquot the required amounts for the students to use each week to minimize contamination. PC12 cells are grown on collagen-coated dishes. The instructor may coat the dishes in advance and provide the students with collagen-coated 6-well plates.

Solutions

Culture medium

Dulbecco’s modified Eagle’s Medium (1 gram/liter glucose) supplemented with 10% fetal calf serum, 5% heat-inactivated donor herd horse serum, 100 units/ml penicillin, and 100 μg/ml streptomycin

Agent stock solutions and concentrations for use

2.5 or 7S NGF

Prepare a 100-μg/ml stock solution and dilute to a final concentration of 100 ng/ml in cell culture medium.

PACAP

Use at 100 nM final concentration in cell culture medium.

Dexamethasone

Prepare a 2.5-mM stock in DMSO and dilute to a final concentration of 2.5 μM in cell culture medium.

Forskolin

Prepare a 10-mM stock in ethanol and dilute to a final concentration of 10 μM in cell culture medium.

Rp-cAMPs

Prepare a 10-mM stock solution and dilute to a final concentration of 10 μM in cell culture medium.

U0126

Prepare a 10-mM stock in DMSO and dilute to a final concentration of 10 μM in cell culture medium.

Phosphate-buffered saline (PBS), pH 8.0

Note: This may be prepared according to standard recipes or purchased.

AChE solubilization buffer

Tris, pH 7.210 mM
NaCl1 M
MgCl250 mM
Triton X-1001%

PCR solubilization buffer

Note: In order to avoid contamination with RNase, it is simplest to extract cellular RNA using a commercially available kit.

Assessment Options: Lab Notebook, Poster, or Journal Article

Students should be required to keep a lab notebook, which can be checked at every lab session or on a schedule of the instructor's choosing. Notebooks should be bound (not loose-leaf) and written in ink, and each entry should include the following information: Date, Title of the Lab, Background, Purpose, Methods, Results, and Conclusions [see (1) for details about how to organize a lab notebook].

One way to teach effective scientific presentation so that the students learn to communicate experiments verbally and visually is to organize a PC12 cell meeting as the last lab session for this series of exercises. Each group should present their results to the class in the form of a poster. The poster should be no larger than 4 feet by 6 feet. The poster should have a title, including the names of all authors. It should also contain the following sections: Abstract, introduction, methods, results, and conclusions [see (1) for details on posters for undergraduate laboratories]. If this lab is combined with labs for assaying AChE activity or the analysis of AChE transcript abundance, then those experiments should be presented all together on the poster. Students may either choose a "spokesperson" to present the poster or may each present a section of the poster to the class. Each student should be able to answer questions about their group's poster.

Another option is to allow each group to submit a journal article on their experiment [see (1) for details about journal articles for undergraduate laboratories]. The article should have an abstract, as well as sections for the introduction, methods, results, and conclusions. The students from other groups can serve as peer-reviewers for articles written by another group, providing constructive comments in writing. The original submission, peer-referees' comments, and the final revised manuscripts will all count toward the final grade for each student.

Information for the Students

Cell Culture as an Experimental Approach

The use of cultured cells grown in vitro has many advantages over the use of an intact animal or an isolated preparation as an experimental approach. This technique permits a far more controlled and easily manipulated cellular environment than can be achieved in an intact animal. There are fewer potential problems with access to the cells of interest and the distribution of pharmacological agents (for instance, there is no need to worry about the blood brain barrier preventing a drug from reaching the neurons under investigation, or the liver metabolizing the substance of interest). Additionally, it is easier to be certain of the exact concentration of an exogenous substance in the vicinity of the cells of interest. Furthermore, the experimenter can selectively alter a single experimental variable and evaluate its effects on a cell with much less concern about possible secondary and tertiary indirect effects from other systems. Because cells in culture continue to grow and divide, unlike isolated experimental preparations, they can be used to study long-term questions about differentiation, development, and the regulation of gene expression. Cell-culture experiments can be broadly divided into two general categories: (i) those that utilize primary cultures and (ii) those that utilize established secondary cell lines.

Primary cultures contain cells that have been harvested from an animal, dissociated, and plated into a culture dish. Because most differentiated cells do not continue to grow and divide indefinitely, most primary cultures do not survive for very long periods of time. Cell lines are cells that have become adapted to living in culture. Although originally isolated from an animal (or a person), they can be grown and propagated in vitro for many years. For example, PC12 cells, which are used in these experiments, are a cell line that has been grown in culture for decades. There are advantages and disadvantages to both approaches. Clonal cell lines (cell lines that are descended from a single original cell) can sometimes provide a more homogeneous source of cell material than do primary cultures and can be grown in more or less unlimited quantities without the need to sacrifice numerous animals. This is a tremendous advantage in doing biochemical analyses. On the other hand, the fact that a cell can be grown indefinitely in culture implies that it has lost some of the differentiated properties of a "normal" cell and become more like a cancer or transformed cell. In fact, many cell lines are derived from spontaneously arising tumors, whereas others arise from cells that have been treated with some transforming agent. Therefore, caution must be used in applying information learned about the properties of cells in secondary cell lines back to the more differentiated cells of the original organism.

PC12 Cells as a Model System

PC12 cells are a secondary cell line that was originally derived from a pheochromocytoma, which is a tumor of the adrenal gland, that developed in an irradiated rat (2). Under ordinary culture conditions, they have properties similar to those of immature rat adrenal chromaffin cells. When grown in the presence of nerve growth factor (NGF), PC12 cells extend neurites, become electrically excitable, become more responsive to exogenously applied acetylcholine, have increased numbers of calcium channels, and increase the synthesis of several neurotransmitters (3). Because PC12 cells grown in the presence of NGF resemble sympathetic neurons, numerous studies have utilized PC12 cells as a readily manipulable, easily grown experimental model for sympathetic neurons. In the presence of the synthetic glucocorticoid dexamethasone, PC12 cells appear to differentiate toward a mature adrenal chromaffin cell-like phenotype with increased activity of the catecholamine synthetic enzyme tyrosine hydroxylase. In many respects, the response of PC12 cells to NGF and dexamethasone is similar to the development of sympathetic neurons and adrenal chromaffin cells from sympathoadrenal precursors (4).

In this series of lab sessions, each group of students will grow PC12 cells in culture and examine the effects of several different agents on differentiation of the cells, which will be assessed recording changes in morphology and cell number: NGF, dexamethasone, PACAP [an adrenomedullary neurotransmitter that promotes neuronal differentiation in PC12 cells (5)], forskolin (a compound that activates adenylate cyclase and hence elevates intracellular levels of the second messenger cAMP), Rp-cAMPs (an inhibitor of cAMP signaling), and U0126 (an inhibitor of the mitogen-activated protein kinase cascade).

Collaboration

These experiments extend over several weeks and involve a collaborative effort on the part of the entire class. Each lab section (Tuesday and Thursday) will be divided into three groups, with each group applying a different combination of agents (Table 1). Because cells in culture are metabolically active, they need to be "fed" (have their nutrient medium removed and replaced) several times a week. Therefore, the Tuesday and Thursday groups will collaborate on feeding cells (for instance, Group One on Tuesday and Group One on Thursday will be collectively responsible for the same culture dishes containing control and treated cells). The instructor will replace the medium over the weekend.

Table 1.

Treatments that influence the differentiation state of PC12 cells.

Guidelines for Cell Culture

Cell culture is not a difficult technique to learn, but it requires care and meticulous attention to detail in order to avoid contaminating the cultures with stray bacteria or fungi. To prevent contamination, the cells are handled in special hoods in which incoming air is filtered, surfaces are decontaminated with 70% ethanol, medium is sterile, and medium, dishes (with or without cells), and other plasticware that will come into contact with the medium should only be opened inside the cell culture hood.

All cell lines grown in culture should be treated with respect. PC12 cells have been used safely for years by numerous laboratories. Thus, PC12 cells are unlikely to pose any danger to the experimenter. However, it is always possible that the particular subline used for these experiments may have been exposed to some pathogen at some time during the past 30 years. The following are important safety precautions that should be followed when working with the cells:

  • Wear gloves when working in the hood.

  • Wipe up any medium spills with 70% ethanol immediately.

  • Dispose of any plasticware (culture dishes, tubes, and pipettes) that has been in contact with the cells in autoclavable biohazard bags.

  • Decontaminate waste medium with a sufficient concentration of bleach to turn the medium clear.

  • Wash hands before leaving the laboratory.

Data

By the end of the 3 weeks, each group in each section will have compiled the following data and shared it with group members in the other lab section:

1. One copy each of the pictures of cells under each of the experimental conditions. (The Tuesday lab sections will have taken six pictures and the Thursday lab sections will have taken 12 pictures. Each group provides pictures to their partner group in the other section, so all students have 18 pictures.)

2. A count of the percent of cells in each picture that have neuritis, and a measurement of the length of the longest neurite.

3. An estimate of the percent confluency of the cells in each picture.

4. The average diameter of 10 representative cells in each picture.

5. The calculated number of cells/well under control and experimental conditions determined from the hemocytometer counts at the time of harvest.

Week One: Demonstration and Practicing Sterile Technique

When a cultured cell line is to be used for experiments, sufficient cells must be available to test all experimental conditions and still have cells available for future experiments. Cultured cells are metabolically active, requiring replenishment of the medium, which supplies the cells' nutrients and accumulates cellular waste products. In addition, the cells divide and will eventually become crowded in the culture dish. Therefore, cultured cells must be passaged (subcultured into new cultures dishes as lower cell density) to allow them to continue to grow in culture. The frequency at which the cells must be passaged and fed depends on several factors: (i) the rate of cell division, (ii) the density of plating, and (iii) the metabolic activity of the cells. Cultured cells do not have to be grown in medium continuously, but can be frozen for long-term storage.

After the instructor demonstrates the techniques for sterile technique and working in a laminar flow hood to feed and passage the cells, each student practices sterile technique. Students should review the guidelines for cell culture prior to working in the hood or counting the cells. Each student will be given a 15-ml sterile plastic tube containing 10 ml Dulbecco's modified Eagle's Medium (do not add serum or antibiotics for this exercise) and asked to perform the following tasks:

1. Label three 15-ml sterile plastic tubes with your name.

2. Pipet 2 ml of medium into each of three sterile test tubes and loosely screw on the caps.

3. Place the tubes in the 37°C incubator to be evaluated for contamination in the next laboratory session.

Week One: Counting Cells Using a Hemocytometer

The density of cells in culture can affect cell proliferation and responsiveness to stimuli; therefore, it is important that each experimental condition be tested on cultures of cells plated at the same density. Moreover, data analysis in this laboratory will involve normalizing enzyme activity to number of cells. Thus, both the number of cells plated into each well at the beginning of the experiment and the number in each well at the end of the experiment must be determined. A hemocytometer is a simple device that is used to count the number of viable cells in a known volume using a microscope (Fig. 1). A hemocytometer contains two equal chambers with a grid pattern of defined size. Each 1-mm square represents a volume of 0.1 mm3 or 10-4 cm3, where 1 cm3 is approximately equal to 1 ml.

Fig. 1.

Counting cells with a hemocytometer. Live cells in the five 1-mm squares highlighted in pale red are counted. Live cells are phase bright (represented as circles with white halos) and dead cells are phase dark and tend to be irregularly shaped (represented by gray irregular shapes).

When preparing cell suspensions for counting, the cells must be evenly dispersed and should be dilute enough that the cells are not overlapping when placed on the hemocytometer. If there are too many clumps of cells or there is excessive unevenness or the density of the cells is so high that many cells are overlapping when placed on the hemocytometer, then clean the hemocytometer and start again with either a more dilute suspension (if too dense and overlapping the first time) or after more effectively and evenly resuspending the cells (if clumpy or uneven the first time).

Viable cells look like phase bright and round; dead cells are phase dark and tend to be irregularly shaped. Only count the live cells. Count the cells in the four corner 1-mm squares and the central 1-mm square (a total of 5 squares). The total count should be at least 50 viable cells per chamber of the hemocytometer. If there are fewer than 50 viable cells after counting the four corners and the central 1-mm square, then count all of the squares. If there are still not 50 viable cells, then the cells must be pelleted by centrifugation and resuspended in a smaller volume, and the count repeated. Some cells may be partially in more than one 1-mm square. Always count these the same way. For example, count a cell if it overlaps the top or right line, and do not count it if it overlaps the bottom or left line. Each group of students will be given a sample of PC12 cells and asked to perform the following tasks:

1. Clean the hemocytometer and coverslip with a small amount of 70% ethanol, and dry with lens paper.

2. Place the cover slip on the hemocytometer.

3. Triturate (gently pipet up and down) the cells to suspend evenly, then using a pasteur pipette, apply a drop of cell suspension to both chambers of the hemocytometer and allow them to fill by capillary action.

4. Starting with the first chamber, count all of the cells in the 1-mm center square and each of the four corner 1-mm squares (Fig. 1).

5. Turn the hemocytometer to the other chamber and count all of the cells in the 1-mm center square and each of the four corner 1-mm squares.

6. Divide by 10 to obtain the average number of cells per 1-mm square. If you counted more than 10 squares because you did not have at least 50 cells after counting one chamber, then divide by the number of squares counted to obtain the average.

7. Calculate the concentration of the cells using the following equation:

$$mathtex$$\[Cells/ml\ =\ Average\ number\ of\ cells\ per\ 1\ mm\ square\ {\times}\ 10^{4}\]$$mathtex$$

Calculate the total number of cells in the sample using the following equation:

$$mathtex$$\[Total\ cells\ =\ (Average\ number\ of\ cells\ per\ 1\ mm\ square\ {\times}\ 10^{4})\ [Volume\ (in\ mls)\ of\ the\ sample]\]$$mathtex$$

Note: Cell viability may also be determined by exclusion of a dye such as Trypan Blue (6). To perform Trypan Blue exclusion, a small volume of cells suspended in PBS is mixed with an equal volume of 0.4% Trypan Blue solution before being placed on the hemocytometer. The additional dilution factor must be accounted for when determining cell number.

Week Two: Tuesday's Lab Section

Depending on the number of students in the lab and access to cell culture hoods, all groups may not be able to work simultaneously. In this case, different groups can be scheduled to arrive in the laboratory at different times.

Group 1, group 2, and group 3 of Tuesday's lab section passages (or "seeds") the cells into 6-well plates. Students should review the general guidelines for sterile technique before starting to seed the cells. Collagen is an extracellular matrix protein that facilitates the growth of neuronally differentiated PC12 cells. Each group receives a 100-mm dish of PC12 cells, three collagen-coated 6-well plates, and the appropriate control and experimental medium containing the various agents. Because forskolin is dissolved in ethanol, and dexamethasone and U0126 are dissolved in DMSO, groups using these agents will need to add appropriate amounts of ethanol or DMSO to the other wells. Cells are plated at a density of 1.3 × 105 cells per well in 2.5 ml of medium. Cells are plated in triplicate for the control and each treatment. Directions are as follows:

1. Visually inspect the cells under the microscope. Record the cells’ appearance and the magnification under which they were observed.

2. Tilt the dish and aspirate the medium off using a pasteur pipette attached to a vacuum line connected to a waste flask. The waste flask should contain bleach in sufficient concentration to remove the color from the medium.

3. Dislodge the cells from dish by vigorously squirting 4 ml of fresh medium onto the cells with a sterile 10-ml pipette. Continue to squirt the medium onto the entire surface of the cell monolayer until all of the cells are in suspension.

Note: Avoid creating too much foam or bubbles by retaining a small volume of medium in the pipette.

4. Triturate the cells to form an even suspension.

5. Count an aliquot of the evenly suspended cells using the hemocytometer.

Note: Each student may count the cells and the average value may be used in the calculations.

6. Calculate the volume of cell suspension (in mls) that will yield 4 × 105 cells total.

7. Place that volume of cell suspension that contains 4 × 105 cells into each of six sterile 15-ml tubes. (Each tube should have 4 × 105 cells.)

8. Add sufficient control medium to the first tube to bring the total volume to 8 ml, and label this tube "C" for control. Add 8 μl of DMSO to this tube. If you are in the group using forskolin, add 8 μl of ethanol.

9. Using a 10-ml pipette, resuspend the cells evenly in the medium and then dispense 2.5 ml into each well of the top row of one of the 6-well plates. Label the plate with your group number (for example, Group 1) and the plate number (#1), the cell type (PC12), the passage number, and the date. Label the top row of wells "control."

10. To the remaining tubes of cells, add sufficient medium containing the appropriate agent (Table 1) to bring the total to 8 ml. Again, make sure to add ethanol or DMSO as appropriate.

11. Using a 10-ml pipette, resuspend the cells evenly in the medium and then dispense 2.5 ml into the wells of the corresponding row of the 6-well plates. Label the plate with your group number (for example, Group 1) and the plate number (#2 and #3), the cell type (PC12), the passage number, and the date. Label each row of wells with the appropriate treatment.

12. Place the tops on the dishes and place the dishes in the 37°C incubator.

13. Wash the bottom of the hood with 70% ethanol and dispose of all used plasticware in the autoclave bag.

14. Remove your gloves and dispose in the autoclave bag, and wash your hands.

15. Draw sketch or a table illustrating what conditions are present in each of the wells of the plates for your group.

Study questions

Why grow the cells on collagen? What purpose does the collagen serve? What might happen if plain culture dishes were used?

Week Two: Thursday's Lab Section

Depending on the number of students in the lab and access to cell culture hoods, all groups may not be able to work simultaneously. In this case, different groups can be scheduled to arrive in the laboratory at different times.

Groups 1, 2, and 3 in Thursday's lab section check the cells that the corresponding group on Tuesday plated for contamination and replace the medium with fresh medium. The students in this lab section also view the cells under the microscope and collect data on the morphology of the cells, noting in particular any differences between the control cells and the cells exposed to the various agents. Photographs of a representative field of cells are taken in order to assess the percent confluence and the average diameter of the cell bodies. If the cells in any of the control or experimental conditions have neurites, the length of the longest neurite in the representative field is also determined. The cells may be viewed under the microscope before replacing the medium or after, although the instructions indicate to view the cells after replacing the medium. Any differences in cell density should also be noted. The students should review the guidelines for sterile technique prior to handling the cells. Detailed instructions are as follows:

1. Examine the wells in each of your group's plates for signs of contamination, which include cloudy, yellow medium or white tufts of material floating on the bottom of the dish. If the well appears contaminated, view the well under microscope for the presence of fungi or bacteria, both of which will be abundant organisms that are smaller than the PC12 cells.

2. If an individual well is contaminated, drain the medium out of that well and then fill the well with 70% ethanol.

3. Drain the medium from the healthy, uncontaminated wells and replace with 2.5 ml of control medium or medium containing the appropriate agent.

4. Drain the ethanol from any contaminated wells.

5. Replace the tops on the plates and observe the cells under the microscope, noting the morphology of the cells and cells’ density in each of the treatment wells and the control wells.

6. Take a photograph of a representative field of cells in a control well and a well representing each of the treatments. Make sure that each student in the group has a copy of the images of the cells. Remember to note the magnification. Measure the length of the longest neurite in the field, measuring from the base of the neurite (where it arises from the cell body) to the tip of the longest filopodium.

7. Replace the plates in the 37°C incubator.

8. Wash the bottom of the hood with 70% ethanol and dispose of all used plasticware in the autoclave bag.

9. Remove your gloves and dispose the autoclave bag, and wash your hands.

Week Three: Tuesday's Lab Section

Depending on the number of students in the lab and access to cell culture hoods, all groups may not be able to work simultaneously. In this case, different groups can be scheduled to arrive in the laboratory at different times.

Groups 1, 2, and 3 in Tuesday's lab section perform the same activities that Thursday's groups performed in week two. They check their group's cells for contamination and replace the medium with fresh medium. They observe the cells under the microscope, noting any changes in morphology and cell density, and obtain an image of the cells. The students follow the same instructions as those for the Thursday groups in week two.

Week Three: Thursday's Lab Section

Thursday's lab section examines the cells under the microscope, noting any morphological differences between the treated cells and the control cells, as well as any differences in cell density. After obtaining an image of the cells, the students harvest the cells. Harvesting is performed outside of the hood on an open lab bench. Each student should harvest the cells from at least one row of their group's 6-well plates and use the hemocytometer to count the cells.

Depending on whether the cells will be used for AChE activity assays or for polymerase chain reaction experiments, the cells are resuspended in one of two buffers at the end of the lab. Details for harvesting the cells are as follows:

1. Observe the cells under the microscope, noting the morphology of the cells and cells’ density in each of the treatment wells and the control wells.

2. Take a photograph of a representative field of cells in a control well and a well representing each of the treatments. Make sure that each student in the group has a copy of the images of the cells. Remember to note the magnification.

3. Label 18 microfuge tubes 1 through 18 with your group number and place them on ice.

4. Remove the medium and the cells and dispose in the waste container with bleach to decontaminate the medium.

Note: If there are three students in a group, then each student can harvest and count the cells from one entire 6-well plate; if there are more than three students, then each student should harvest and count the cells representing the control or one set of the treated cells.

5. Rinse the cells three times with phosphate-buffered saline by gently applying the solution to the side of the well so that the cells are not dislodged and gently removing the solution with a pipette after each wash. Discard each wash in the bleach-containing waste container.

6. Resuspend the cells in each well in 1 ml of phosphate-buffered saline. Try to resuspend them by squirting them vigorously with the phosphate-buffered saline. If there are still cells attached to the plate, then scrape them off of the plate with a rubber policeman.

7. Place the cells from each well in the corresponding microfuge tube on ice.

8. Remove a drop of cell suspension from one well of the control cells and each experimental treatment group, and count the cells with the hemocytometer.

9. Spin the tubes for 1 min at maximum speed in a microcentrifuge at room temperature.

10. Gently aspirate the supernatant without dislodging the cell pellet. Discard the supernatant in the bleach-containing waste container.

11a. Resuspend the cells in 500 μl of ice-cold solubilization buffer and store at −80°C for use in the AChE activity laboratory.

11b. Resuspend the cells in the appropriate solution and store at −80°C for use in the PCR laboratory.

Study questions

Why can the cells be harvested outside of the hood? Why is sterility not essential anymore?

Educational Details

Learning Resource Type: Laboratory exercise

Context: Undergraduate upper division

Intended Users: Teacher

Intended Educational Use: Teach, plan, assess

Discipline: Neurobiology, pharmacology, cell biology

Keywords: Signal transduction, cell culture, chromaffin cell, neuron

Related Resources

Connections Map

D. Vaudry, P. J. S. Stork, P. Lazarovici, L. E. Eiden, Differentiation pathway in PC12 cells. Sci. STKE (Connections Map, as seen September 2006), About Connections Map.

Teaching Resource

E. M. Adler, Cell culture as a model system for teaching: Using PC12 cells. Sci. STKE 2006, tr5. [Abstract] [Full Text]

References

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