Protocols

The following protocols are general procedures that can be used as templates to construct a specific protocol for an experimental assay.  Investigators are strongly encouraged to collaborate with the facility to ensure that appropriate procedures with proper controls are implemented.

 

INDIRECT IMMUNOFLUORESCENCE STAINING  

DIRECT IMMUNOFLUORESCNCE STAINING  

REAGENTS

 

 

INDIRECT STAINING PROCEDURE

 

Materials

  1. Cells (~20 x 106/ml)
  2. Monoclonal antibody
  3. Anti-IgG FITC conjugate
  4. Wash solution (1X Dulbecco's PBS, Ca2+ and Mg2+ free)
  5. 1% formaldehyde-PBS (2% stock solution diluted with 1X  PBS)

 

Method

  1. Add 50 µl of cells to each 12 x 75 mm polystyrene Falcon tube (Becton Dickinson #2054, University Stores).  The cells should be adjusted to approximately 20 x 106 cells per ml (50 µl = 1 x 106 cells per tube).  Add appropriate volume of monoclonal antibody to the test sample tubes.  Include one tube for an autofluorescence control sample and another tube for secondary antibody fluorochrome conjugate control.  If needed include an isotype control.  Add the same volume of 1X PBS to the autofluorescence and secondary antibody fluorochrome conjugate control tubes.  MIX BY VORTEXING ALL SAMPLES.
  2. Incubate for 30 minutes at room temperature (or appropriate temperature for cells) in the dark.
  3. Note: If the incubation temperatures are at 4oC, then the centrifugation washes should be performed at 4oC. Likewise, if incubations are at room temperature (RT), then centrifugation is carried out at RT. Generally, staining of leukocytes from peripheral whole blood followed by subsequent red cells lysis is performed at RT. Leukocytes derived from gradients and cultures are kept at 4oC during staining procedures.
  4. Wash cells in Falcon tubes with 3 ml volumes of 1X PBS and centrifuge samples at 200 x g (1000 rpm's) for 10 minutes at room temperature. Decant supernatant fluid.  Add appropriate volume of diluted anti-IgG fluorochrome conjugate to the antibody samples tubes and secondary antibody control tube.  Add the same volume of 1X PBS to the autofluorescence control tube.  MIX BY VORTEXING ALL SAMPLES.
  5. Incubate for 30 minutes at room temperature (or appropriate temperature for the cells) in the dark.
  6. Wash cells in Falcon tubes with 3 ml volumes of 1X PBS and centrifuge samples at 200 x g (1000 rpm's) for 10 minutes at room temperature.  Decant supernatant fluid.  VORTEX the cell pellet in the residual wash fluid and add 300 µl of 1% formaldehyde-PBS to each sample and quickly VORTEX again. (Be sure to vortex prior to adding fixative or you will subsequently fix a pellet and not a single cell suspension).
  7. Store samples at 1-6oC in the dark until FACS analysis.

Reference:  Current Protocols in Immunology.  Chapter 5. pp. 7.9.1 - 7.9.2.  1991.

 

 

DIRECT STAINING PROCEDURE

 

Materials 

  1. Cells (~20 x 106/ml)
  2. Directly conjugated monoclonal antibody
  3. Wash solution (1X Dulbecco's PBS, Ca2+ and Mg2+ free)
  4. 1% formaldehyde-PBS (2% stock solution diluted with 1X  PBS)

 

Method

  1. Add 50 µl of cells to a 12 x 75 mm polystyrene Falcon tube (Becton Dickinson #2054, University Stores).  The cells should be adjusted to approximately 20 x 106 cells per ml (50 µl = 1 x 106 cells per tube).  Add appropriate volume of fluorochrome conjugated monoclonal antibody to test sample tubes.  Include one tube for an autofluorescence control sample and another tube for an isotype control tube.  Add the same volume of 1X PBS to the autofluorescence control tube.  MIX BY VORTEXING ALL SAMPLES.
  2. Incubate for 20-30 minutes at room temperature (or appropriate temperature for cells) in the dark.
  3. Note: If the incubation temperatures are at 4oC, then the centrifugation washes should be performed at 4oC. Likewise, if incubations are at room temperature (RT), then centrifugation is carried out at RT. Generally, staining of leukocytes from peripheral whole blood followed by subsequent red cells lysis is performed at RT. Leukocytes derived from gradients and cultures are kept at 4oC during staining procedures.
  4. Wash cells in Falcon tubes with 3 ml volumes of 1X PBS and centrifuge samples at 200 x g (1000 rpm's) for 10 minutes at room temperature. Decant supernatant fluid.  
  5.  VORTEX the pellet in the residual wash fluid and add 300 µl of 1% formaldehyde-PBS to each sample and quickly VORTEX again.  (Make sure you vortex prior to adding the fixative or you will subsequently fix a pellet and not a single cell suspension).
  6. Store samples at 1-6oC in the dark until FACS analysis.

Reference:  Current Protocols in Immunology.  Chapter 5. pp. 7.9.1 - 7.9.2.  1991

 

REAGENTS

 

1X Ammonium Chloride Lysing Buffer

 

0.829 g  NH4Cl

0.109 g  KHCO3

0.0037g  Disodium EDTA

 

QS to 100 ml with glass distilled water.  Adjust  pH to 7.3 to 7.4.  Store at room temperature and use within 24 hours.

 

Formaldehyde Fixative

 

A 2% formaldehyde stock solution is made by adding 2.0 g paraformaldehyde to 100 ml of 1X PBS (Ca2+ and Mg2+ free).  Heat to 70oC in a fume hood until the paraformaldehyde goes into solution.  Allow the solution to cool to room temperature.  Filter through a 0.45 µm filter.  Adjust to pH 7.2 - 7.4 using 1 M NaOH or 1 M HCl as needed.  Store in the refrigerator.

 

Reference: Jackson, A.L. and Warner, N.L.  Manual of Clinical Laboratory Immunology.  Chapter 32.  226-235.  1986.