Other equipment:
The RCL also houses a 6-foot laminar flow tissue culture hood, a Shandon Cytospin II cytocentrifuge, water bath, pH meter, fluorescence microscope,refrigerator, water-jacketed CO2 incubator, and clinical centrifuge.

Other equipment:
The
RCL also houses a 6-foot laminar flow tissue culture hood, a Shandon Cytospin II cytocentrifuge, water bath, pH meter, fluorescence microscope,refrigerator, water-jacketed CO2 incubator, and clinical centrifuge.

Sample Requirements

Analysis

•·     Cells must be in suspension, either in PBS or tissue culture medium.

•·     Cell concentration should be from 0.5 to 1 x 10E6 cells/ml with a minimum volume of 0.2 ml (minimum of 1 to 2x105 cells).

•·     It is important that you do a cell count on your sample before bringing them to the FACS lab it is important that you know how many cells you have.

•·     Cell samples should be in 6-ml, polypropylene, round bottom tubes (Falcon #2063). Other brands of round bottom tubes may appear to be the same as the Falcon 2063 tubes but they are not (many have a smaller inner diameter). Be sure your samples are in the correct tubes otherwise they won't fit on the FACS sample pickup line and time will be wasted in transferring them to the proper tubes.

•·     Samples should be free of cell clumps and debris.

•·     Viability of cell sample should be high, preferably =/> 90%

•·     It is also important to include positive and negative controls if possible.

•·     A typical experiment might contain the following sample:

Negative controls
Cells without any added antibody (autofluorescence)
Cells plus a non-reactive antibody (non-specific binding control, also called isotype control)

Positive control
Cells plus an antibody known to bind to the cells being used.

Experimental
Cells plus antibody in question (including perhaps several dilutions)

Other controls that are sometimes included
Cells that would not be expected to bind the experimental antibody being tested. For example, if the antibody being tested is reactive with malignant cells, you may want to include a "normal" cell to rule out non-specific binding.

Sorting

•·     Cells must be in suspension.

•·     Cells should be at a concentration of 5 to 10 x 10E6/ml. The total number of cells required will depend on the number of sorted (purified) cells you require the percentage represented by the cells of interest.

•·     For example, if the cell you wish to sort represents 10% of the total cells and you require 1 x 10E7 purified (sorted) cells, you would need to bring 1 x 108 stained cells to the FACS lab assuming 100% recovery, which is never the case. For example,

•·     Typical Sort Recoveries on the FACSVantage

Flow Rate (cells/sec)

1000

2000

5000

10000

Recovery (*)

95%

90%

70%

60%

Purity (**)

99%

99%

99%

99%

(*) the percentage of positive cells actually recovered during the sort.
(**) the percentage of the recovered cells that are positive for the marker under investigation [CD4 in the example below].

Example of sort recovery:
A sample of 36 x 10E6 cells is brought to the FACS lab after being stained with a CD4 antibody; FACS analysis indicates the sample has 35% fluorescence-positive (CD4+) cells in it. The sample requires 1 hr of sorting time (at 10,000 cells/sec). At 36% positivity, the theoretical yield of CD4 cells would be 12 x 10E6 cells: [(36 x 10E6) x (0.35)] At a recovery rate of 65%, we are able to collect 8 x 10E6 cells [(12 x 10E6) x (0.65)] which will be =/>99% pure, i.e., 99% of them will be CD4+.

Links:

•·     International Society of Analytical Cytology (ISAC) Home Page Society information, news, journals, and other links.

•·     Join the Cytometry Mailing List. Also, lists archived messages.

•·     Univ. of Massachusetts, Amherst. Catalogue of free flow cytometry software including MFI; flow cytometry explanation.

•·     Molecular Probes. Fluorescent probes and other flow cytometry and fluorescence microscopy reagents.

•·     Cytometry Servers List at Purdue. Websites, scientific societies, and online journals.

•·     Beckman-Coulter. Antibodies and other flow cytometry reagents.

back to top



Cytocentrifugation

General Information

The Shandon Cytospin was the first device to use low-speed centrifugal force to separate and deposit a monolayer of cells on slides while maintaining cellular integrity.

Sample Requirements

Cells must be in suspension.

Since it is difficult to determine how many cells will be required to produce a monolayer on the slide, several dilutions of the sample are usually performed. Since only a few hundred cells are required for most microscopic observations, the initial number of cells required is low, e.g., 5,000 to 10,000/ml in a total volume of 0.5 to 1.0ml.

Cells may be diluted in tissue culture medium, PBS, etc.

From 0.2 to 0.4 ml of cells is used for each cell dilution.

After centrifuging for approximately 5 min, the deposited cells are ready for fixation followed by subsequent staining.

Cells may be fixed and stained prior to centrifuging.

User Fees:
Please contact Dr. Raymond Hester at (251) 460-6029.

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Techniques and Protocols

Ploidy (DNA) Cell Cycle Analysis Using Propidium Iodide

Technical Points

•1.  It is often necessary to try more than one technique before finding the one that will work best with your cells.

•2.  Some cell types tend to clump during the propidium iodide staining procedure. If cell clumps are present, there is a good possibility that the cells cannot be analyzed since they will clog the sample intake line of the flow cytometer.

•3.  You should always examine at least one of your sample tubes prior to bringing them to the FACS lab to see if clumping is present. The easiest way to do this is with a hemacytometer and low power (10x or 20x) objective of a microscope.

•4.  Please have your samples in Falcon #2063 tubes when you bring them to the lab. THESE ARE THE ONLY TUBES THAT WILL WORK ON THE FACS.

•5.  If the cells you are working with are adherent cells and you are using trypsin to detach them from culture dishes, you should not let these cells stay in the presence of trypsin for any length of time longer than necessary.

Procedure A (original technique as reported by Krishan, plus a modification)

Krishan introduced ploidy analysis using propidium iodide. Later his technique was modified by the addition of NP-40. Procedures for both the original and the modification are described below. Krishan Procedure [*]

•1.  Resuspend 1 x 10 6 cells in 1 ml of P.I. solution (0.05 mg/ml propodium iodide in 0.1% sodium citrate)

•2.  Incubate 5 to 10 min at 4 degrees C

•3.  Analyze by flow cytometry

Modified Krishan Precedure [**]

•1.  Resuspend 1 x 10 6 cells in 1 ml of modified Krishan staining buffer; vortex 10 secs.

•2.  Incubate at least 30 minutes on ice; centrifuge

•3.  Resuspend pellet in 1 ml fresh modified Krishan staining buffer

•4.  Syringe through 27g needle

•5.  Filter through 37 micron nylon mesh immediately before flow analysis

Modified Krishan buffer

•·     0.1% sodium citrate

•·     0.3% NP-40

•·     0.05 mg/ml propidium iodide

•·     0.02 mg/ml RNase

References

[*] A. Krishan, Rapid cytoflurorometric analysis of mammalian cell cycle by propidium iodide staining, J. Cell. Biol. 66:188-193.

[**] L.G. Dressler, et. al., 1988, DNA flow cytometry and prognostic factors in 1331 frozen breast cancer specimens, Cancer 61:420-427.

Procedure B (Vindalov modification - has been used with human carcinoma cell lines)

The following procedure has been used successfully by Gudrun Urban (Biochemistry) for cell cycle analysis of A549 (lung carcinoma) and MC-F-7 (breast carcinoma) cells.

•·     Remove media and non-adherent cells from culture dish (and save if they are to be analyzed).

•·     Remove adherent cells from culture dish with trypsin being careful that the cells are harvested as soon as they begin detaching from the dish; do not leave them in trypsin any longer than necessary.

•·     Transfer trypsin-treated cells to conical centrifuge tubes.

•·     Centrifuge, 5 min, 1,000 rpm.

•·     Pour off supernate and resuspend pellet in 1 ml PBS+PI buffer.

•·     Perform cell count using a hemacytometer.

•·     Centrifuge cells again, 5 min, 1,000 rpm.

•·     Pour off supernate and resuspend cells in PBS+PI (50 ug/ml) to a concentration of 1 x 106 cells/ml.

•·     Transfer 1 ml of cell suspension to a 15-ml conical centrifuge tube.

•·     Add 1 ml (equal volume) Vindelov's PI to each tube.

•·     Incubate 1 hr at 4C in the dark.

•·     Transfer to Falcon 2063 tubes for FACS analysis. May be kept at 4C O.N.

Reagents

PBS + PI

filtered PBS + PI (50µg/ml)

Vindelov's Propidium Iodide Stain

 

1.21 g Tris base (0.01 M)

50.1 mg propidium iodide (7.5x10 -5 M)

584 mg NaCl (10mM)

1.0 ml Igepal (equiv to Np-40)

10 mg Rnase (700 U)

 

This recipe is per 1 liter. Adjust pH to 8.0 and filter through a 0.2µ filter. Wrap bottle in aluminum foil and store at 4C in the dark. Discard after one month.

[Adapted from DNA Cell Cycle Analysis, Propodium iodide procedute, Handbook of Flow Cytometry Methods, J. Paul Robinson, Ed., Wiley Liss, 1993. This article further references, Krishan, A., Rapid flow cytometric analysis of mammalian cell cycle by propidium iodide staining, J. Cell Biol., 66:188-193, 1975]

Procedure C (low pH modification - has been used with endothelial cells)

The following procedure has been used successfully by David Killilea (Pharmacology) for cell cycle analysis of rat pulmonary artery smooth muscle cells (RPA-SMC)

•·     Grow up cell cultures and expose to appropriate experimental conditions.

•·     Trypsinize (0.25%, GIBCO) cells and collect in 15-ml conical tubes. At this point, many procedures call for the addition of buffer with serum to inactivate the trypsn; RPA-SMC apparently do not require this step.

•·     Centrifuge 5 min at 1,500 rpm.

•·     Decant supernate and resuspend in 1 ml of PBS (pH 8).

•·     Remove 30µl from each sample and place in microfuge tubes with 30µl of 0.4% trypan blue. Mix gently.

•·     Count viable cells using a hemacytometer and calculate # cells/ml.

•·     Transfer volume of cell suspension necessary for 1-5 x 106 cells to a centrifuge tube (microfuge, if possible).

•·     Centrifuge 5 min at 1,500 rpm.

•·     Decant supernate and resuspend in 150µl of 1x PBS (pH8).

•·     Add 375 µl of FACS buffer to each tube. Mix.

•·     Add 250µl of RNAse to each tube.

•·     Add 100µl of 0.2% Triton-X 100 to each tube. Mix.

•·     Incubate at 37C in 5% CO2 in air for 30 min.

•·     Add 100 µl of propidium iodide solution to each tube. Cover with aluminum foil.

•·     Incubate at R.T. for 30 min.

•·     Transfer cell suspensions to Falcon 2063 tubes for FACS analysis. Samples may be stored O.N. at 4C.

Reagents

FACS buffer: (250 ml: store at R.T.)

 

100 mM sodium acetate

3.4 g NaC 2 H 3 O 2

5.4 mM EDTA

+0.5 g EDTA

pH to 5.2

 

qs 250 ml

 

 

 

RNAse: (for 1 ml; prepare fresh)

 

700 U/ml RNAse

10 mg of 70 U/mg RNAse

 

+1 ml FACS buffer

 

 

0.2% Triton-X 100: (for 50 ml)

1 ml undiluted Trition-X 100

 

+49 ml FACS buffer

 

 

propodium iodide solution: (for 2ml)

1 mg propodium iodide

 

+ 2 ml FACS buffer

[Adapted from R.L. Vendor, et. al., Reduced oxygen tension induces pulmonary endothelium to release a pulmonary smooth muscle cell mitogen(s), Amer. Rev. Resp. Dis., 135:622-7, 1987.]

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•·      

Sample Requirements

Analysis

•·     Cells must be in suspension, either in PBS or tissue culture medium.

•·     Cell concentration should be from 0.5 to 1 x 10E6 cells/ml with a minimum volume of 0.2 ml (minimum of 1 to 2x105 cells).

•·     It is important that you do a cell count on your sample before bringing them to the FACS lab it is important that you know how many cells you have.

•·     Cell samples should be in 6-ml, polypropylene, round bottom tubes (Falcon #2063). Other brands of round bottom tubes may appear to be the same as the Falcon 2063 tubes but they are not (many have a smaller inner diameter). Be sure your samples are in the correct tubes otherwise they won't fit on the FACS sample pickup line and time will be wasted in transferring them to the proper tubes.

•·     Samples should be free of cell clumps and debris.

•·     Viability of cell sample should be high, preferably =/> 90%

•·     It is also important to include positive and negative controls if possible.

•·     A typical experiment might contain the following sample:

Negative controls
Cells without any added antibody (autofluorescence)
Cells plus a non-reactive antibody (non-specific binding control, also called isotype control)

Positive control
Cells plus an antibody known to bind to the cells being used.

Experimental
Cells plus antibody in question (including perhaps several dilutions)

Other controls that are sometimes included
Cells that would not be expected to bind the experimental antibody being tested. For example, if the antibody being tested is reactive with malignant cells, you may want to include a "normal" cell to rule out non-specific binding.

Sorting

•·     Cells must be in suspension.

•·     Cells should be at a concentration of 5 to 10 x 10E6/ml. The total number of cells required will depend on the number of sorted (purified) cells you require the percentage represented by the cells of interest.

•·     For example, if the cell you wish to sort represents 10% of the total cells and you require 1 x 10E7 purified (sorted) cells, you would need to bring 1 x 108 stained cells to the FACS lab assuming 100% recovery, which is never the case. For example,

•·     Typical Sort Recoveries on the FACSVantage

Flow Rate (cells/sec)

1000

2000

5000

10000

Recovery (*)

95%

90%

70%

60%

Purity (**)

99%

99%

99%

99%

(*) the percentage of positive cells actually recovered during the sort.
(**) the percentage of the recovered cells that are positive for the marker under investigation [CD4 in the example below].

Example of sort recovery:
A sample of 36 x 10E6 cells is brought to the FACS lab after being stained with a CD4 antibody; FACS analysis indicates the sample has 35% fluorescence-positive (CD4+) cells in it. The sample requires 1 hr of sorting time (at 10,000 cells/sec). At 36% positivity, the theoretical yield of CD4 cells would be 12 x 10E6 cells: [(36 x 10E6) x (0.35)] At a recovery rate of 65%, we are able to collect 8 x 10E6 cells [(12 x 10E6) x (0.65)] which will be =/>99% pure, i.e., 99% of them will be CD4+.

Links:

•·     International Society of Analytical Cytology (ISAC) Home Page Society information, news, journals, and other links.

•·     Join the Cytometry Mailing List. Also, lists archived messages.

•·     Univ. of Massachusetts, Amherst. Catalogue of free flow cytometry software including MFI; flow cytometry explanation.

•·     Molecular Probes. Fluorescent probes and other flow cytometry and fluorescence microscopy reagents.

•·     Cytometry Servers List at Purdue. Websites, scientific societies, and online journals.

•·     Beckman-Coulter. Antibodies and other flow cytometry reagents.

back to top



Cytocentrifugation

General Information

The Shandon Cytospin was the first device to use low-speed centrifugal force to separate and deposit a monolayer of cells on slides while maintaining cellular integrity.

Sample Requirements

Cells must be in suspension.

Since it is difficult to determine how many cells will be required to produce a monolayer on the slide, several dilutions of the sample are usually performed. Since only a few hundred cells are required for most microscopic observations, the initial number of cells required is low, e.g., 5,000 to 10,000/ml in a total volume of 0.5 to 1.0ml.

Cells may be diluted in tissue culture medium, PBS, etc.

From 0.2 to 0.4 ml of cells is used for each cell dilution.

After centrifuging for approximately 5 min, the deposited cells are ready for fixation followed by subsequent staining.

Cells may be fixed and stained prior to centrifuging.

User Fees:
Please contact Dr. Raymond Hester at (251) 460-6029.

back to top


Techniques and Protocols

Ploidy (DNA) Cell Cycle Analysis Using Propidium Iodide

Technical Points

•1.  It is often necessary to try more than one technique before finding the one that will work best with your cells.

•2.  Some cell types tend to clump during the propidium iodide staining procedure. If cell clumps are present, there is a good possibility that the cells cannot be analyzed since they will clog the sample intake line of the flow cytometer.

•3.  You should always examine at least one of your sample tubes prior to bringing them to the FACS lab to see if clumping is present. The easiest way to do this is with a hemacytometer and low power (10x or 20x) objective of a microscope.

•4.  Please have your samples in Falcon #2063 tubes when you bring them to the lab. THESE ARE THE ONLY TUBES THAT WILL WORK ON THE FACS.

•5.  If the cells you are working with are adherent cells and you are using trypsin to detach them from culture dishes, you should not let these cells stay in the presence of trypsin for any length of time longer than necessary.

Procedure A (original technique as reported by Krishan, plus a modification)

Krishan introduced ploidy analysis using propidium iodide. Later his technique was modified by the addition of NP-40. Procedures for both the original and the modification are described below. Krishan Procedure [*]

•1.  Resuspend 1 x 10 6 cells in 1 ml of P.I. solution (0.05 mg/ml propodium iodide in 0.1% sodium citrate)

•2.  Incubate 5 to 10 min at 4 degrees C

•3.  Analyze by flow cytometry

Modified Krishan Precedure [**]

•1.  Resuspend 1 x 10 6 cells in 1 ml of modified Krishan staining buffer; vortex 10 secs.

•2.  Incubate at least 30 minutes on ice; centrifuge

•3.  Resuspend pellet in 1 ml fresh modified Krishan staining buffer

•4.  Syringe through 27g needle

•5.  Filter through 37 micron nylon mesh immediately before flow analysis

Modified Krishan buffer

•·     0.1% sodium citrate

•·     0.3% NP-40

•·     0.05 mg/ml propidium iodide

•·     0.02 mg/ml RNase

References

[*] A. Krishan, Rapid cytoflurorometric analysis of mammalian cell cycle by propidium iodide staining, J. Cell. Biol. 66:188-193.

[**] L.G. Dressler, et. al., 1988, DNA flow cytometry and prognostic factors in 1331 frozen breast cancer specimens, Cancer 61:420-427.

Procedure B (Vindalov modification - has been used with human carcinoma cell lines)

The following procedure has been used successfully by Gudrun Urban (Biochemistry) for cell cycle analysis of A549 (lung carcinoma) and MC-F-7 (breast carcinoma) cells.

•·     Remove media and non-adherent cells from culture dish (and save if they are to be analyzed).

•·     Remove adherent cells from culture dish with trypsin being careful that the cells are harvested as soon as they begin detaching from the dish; do not leave them in trypsin any longer than necessary.

•·     Transfer trypsin-treated cells to conical centrifuge tubes.

•·     Centrifuge, 5 min, 1,000 rpm.

•·     Pour off supernate and resuspend pellet in 1 ml PBS+PI buffer.

•·     Perform cell count using a hemacytometer.

•·     Centrifuge cells again, 5 min, 1,000 rpm.

•·     Pour off supernate and resuspend cells in PBS+PI (50 ug/ml) to a concentration of 1 x 106 cells/ml.

•·     Transfer 1 ml of cell suspension to a 15-ml conical centrifuge tube.

•·     Add 1 ml (equal volume) Vindelov's PI to each tube.

•·     Incubate 1 hr at 4C in the dark.

•·     Transfer to Falcon 2063 tubes for FACS analysis. May be kept at 4C O.N.

Reagents

PBS + PI

filtered PBS + PI (50µg/ml)

Vindelov's Propidium Iodide Stain

 

1.21 g Tris base (0.01 M)

50.1 mg propidium iodide (7.5x10 -5 M)

584 mg NaCl (10mM)

1.0 ml Igepal (equiv to Np-40)

10 mg Rnase (700 U)

 

This recipe is per 1 liter. Adjust pH to 8.0 and filter through a 0.2µ filter. Wrap bottle in aluminum foil and store at 4C in the dark. Discard after one month.

[Adapted from DNA Cell Cycle Analysis, Propodium iodide procedute, Handbook of Flow Cytometry Methods, J. Paul Robinson, Ed., Wiley Liss, 1993. This article further references, Krishan, A., Rapid flow cytometric analysis of mammalian cell cycle by propidium iodide staining, J. Cell Biol., 66:188-193, 1975]

Procedure C (low pH modification - has been used with endothelial cells)

The following procedure has been used successfully by David Killilea (Pharmacology) for cell cycle analysis of rat pulmonary artery smooth muscle cells (RPA-SMC)

•·     Grow up cell cultures and expose to appropriate experimental conditions.

•·     Trypsinize (0.25%, GIBCO) cells and collect in 15-ml conical tubes. At this point, many procedures call for the addition of buffer with serum to inactivate the trypsn; RPA-SMC apparently do not require this step.

•·     Centrifuge 5 min at 1,500 rpm.

•·     Decant supernate and resuspend in 1 ml of PBS (pH 8).

•·     Remove 30µl from each sample and place in microfuge tubes with 30µl of 0.4% trypan blue. Mix gently.

•·     Count viable cells using a hemacytometer and calculate # cells/ml.

•·     Transfer volume of cell suspension necessary for 1-5 x 106 cells to a centrifuge tube (microfuge, if possible).

•·     Centrifuge 5 min at 1,500 rpm.

•·     Decant supernate and resuspend in 150µl of 1x PBS (pH8).

•·     Add 375 µl of FACS buffer to each tube. Mix.

•·     Add 250µl of RNAse to each tube.

•·     Add 100µl of 0.2% Triton-X 100 to each tube. Mix.

•·     Incubate at 37C in 5% CO2 in air for 30 min.

•·     Add 100 µl of propidium iodide solution to each tube. Cover with aluminum foil.

•·     Incubate at R.T. for 30 min.

•·     Transfer cell suspensions to Falcon 2063 tubes for FACS analysis. Samples may be stored O.N. at 4C.

Reagents

FACS buffer: (250 ml: store at R.T.)

 

100 mM sodium acetate

3.4 g NaC 2 H 3 O 2

5.4 mM EDTA

+0.5 g EDTA

pH to 5.2

 

qs 250 ml

 

 

 

RNAse: (for 1 ml; prepare fresh)

 

700 U/ml RNAse

10 mg of 70 U/mg RNAse

 

+1 ml FACS buffer

 

 

0.2% Triton-X 100: (for 50 ml)

1 ml undiluted Trition-X 100

 

+49 ml FACS buffer

 

 

propodium iodide solution: (for 2ml)

1 mg propodium iodide

 

+ 2 ml FACS buffer

[Adapted from R.L. Vendor, et. al., Reduced oxygen tension induces pulmonary endothelium to release a pulmonary smooth muscle cell mitogen(s), Amer. Rev. Resp. Dis., 135:622-7, 1987.]

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