Lab Protocols

MATERIALS: 1. 50 mg vial Indo-1 (Cat # I-1203, Molecular Probes, OR) 2. DMSO (Sigma, St. Louis, MO) 3. RPMI 1640 4. Monoclonal antibodies (mAb), conjugated to suitable fluorochromes 5. Ionomycin (Calbiochem, San Diego, CA) 6. 37°C water bath, centrifuge, vortexer. 7. Agonists to test Ca2+ flux, e.g. anti-CD3, anti-IgG, ConA. 8. Serum (for RPMI with 2% serum, if cells require serum). METHOD: 1. Incubate cells (£2x107/ml) in RPMI with 1-5 mM Indo-1 (acetoxymethyl ester) at 37°C for 40 min for loading. 2. Incubate aliquots of Indo-1 loaded cells with saturating concentrations of e.g., FITC, PE, PerCP, or Tricolor-conjugated antibodies for 20 min. Incubate at 20° to 25oC unless the antigen is subject to capping, otherwise use 4° to 8oC. Note: mAbs must be azide free. Note: set-up single color stained cells for setting appropriate fluorescence compensation on the instrument. 3. Wash cells twice in RPMI and suspend them at the desired concentration (usually 2x106/ml...

Materials Phosphate buffered saline (1 X PBS without Ca++ and Mg++) Newborn calf serum (NCS) Sodium azide (NaAz) Nucleic acid staining solution (NASS, phosphate-citrate buffer tablets, sodium chloride, sodium ethylene-diaminetetraacetic acid (EDTA), bovine serum albumin (BSA), all from Sigma), see recipe Dimethylsulfoxide (DMSO) Saponin (Sigma) 7-amino-actinomycin D (7-AAD) stock solution, see recipe Actinomycin D (C1) (AD, Roche Molecular Biosystems) stock solution, see recipe Method 1. Place 1 x 106 PBS-washed cells into a 12 x 75 mm tube and add 100 µl of PBS supplemented with 2% NCS and 0.1% NaAz and mix well. 2. For staining of cell surface antigen expression add appropriate amounts of biotinylated, phycoerythrin (PE)-labelled and allophycocyanin (APC)-labelled monoclonal antibodies (mAb) or of corresponding labelled isotypic control antibody and incubate the samples while protected from light for 15 min at 20oC - 25oC. 3. Wash cells once with 2 ml of 1 X PBS by centri...

Materials Phosphate buffered saline (1 X PBS without Ca++ and Mg++) Newborn calf serum (NCS) Sodium azide (NaAz) Nucleic acid staining solution (NASS, phosphate-citrate buffer tablets, sodium chloride, sodium ethylene-diaminetetraacetic acid (EDTA), bovine serum albumin (BSA), all from Sigma), see recipe Dimethylsulfoxide (DMSO) Saponin (Sigma) 7-amino-actinomycin D (7-AAD) stock solution, see recipe Pyronin Y(G) (PY) (Polysciences) Actinomycin D (C1) (AD, Roche Molecular Biosystems) stock solution, see recipe Method Place 1 x 106 PBS-washed cells into a 12 x 75 mm tube and add 100 µl of PBS supplemented with 2% NCS and 0.1% NaAz and mix well. For staining of cell surface antigen expression add appropriate amounts of biotinylated and allophycocyanin (APC)-labelled monoclonal antibodies (mAb) or of corresponding labelled isotypic control antibody and incubate the samples while protected from light for 15 min at 20ºC - 25ºC. Wash cells once with 2 ml of 1 X PBS ...

Materials Phosphate buffered saline (1 X PBS without Ca++ and Mg++)Newborn calf serum (NCS) Sodium azide (NaAz) Nucleic acid staining solution (NASS, phosphate-citrate buffer tablets, sodium chloride, sodium ethylene-diaminetetraacetic acid (EDTA), bovine serum albumin (BSA), all from Sigma), see recipe Ribonuclease A (RNAse) Dimethylsulfoxide (DMSO) Saponin (Sigma)   7-amino-actinomycin D (7-AAD) stock solution, see recipe TO-PRO-3 iodide (TP3) (Molecular Probes), note that TP3 requires a flow cytometer capable of red excitation, e.g., 633 nm, 635 nm Actinomycin D (C 1 ) (AD, Roche Molecular Biosystems) stock solution, see recipeµ Method   Place 1 x 10 6 PBS-washed cells into a 12 x 75 mm tube and add 250 µl of PBS supplemented with 2% NCS and 0.1% NaAz and containing 4 µg/ml of 7-AAD and mix well. For staining of cell surface antigen expression add appropriate amounts of FITC-labeled and PE-labeled monoclonal antibodies (mAb) or of FITC...

Reagents Cells to be studied expressing green fluorescent protein (GFP). Note that the same cell type without GFP is needed as control. Hoechst 33342 stock solution (1mg/ml) (see recipe) 12 X 75 mm culture tubes Vortex mixer Waterbath at 37oC Method 1. Count cells. 2. Place approximately 106 cells into a 12 x 15 mm test tube and spin them down by centrifugation for 5 min at 300 x g. 3. Remove supernatant by aspiration or rapid decanting and add 500 ml of the medium that was used for growing the cells to be studied pre-warmed to 37oC to the cell pellet. Mix gently. Add 5 ml of Hoechst 33342 stock solution and mix again. Incubate at 37oC for 45 min. The optimal Hoechst dye concentration and staining time for different cell types vary as dye up-take depends on cellular metabolic rates; thus, both have to be determined empirically. In general, dye concentrations between 1mg/ml and 10 mg/ml and incubation times between 20 min and 90 min will produce DNA histograms with ac...

Reagents Cells to be studied expressing green fluorescent protein (GFP). Note that the same cell type without GFP is needed as a control. 1 X PBS 2% Buffered formaldehyde solution (see recipe) 70% Ethanol Propidium iodide stock solution (1mg/ml in PBS) DNAse-free ribonuclease A 2 X 75 mm culture tubes Vortex mixer Waterbath at 37oC Method Fix cells with formaldehyde 1. Count cells. 2. Place approximately 106 cells into a 12 x 15 mm test tube and wash them once with PBS by centrifugation for 5 min at 300 x g at 2-8oC. 3. Remove supernatant by aspiration or rapid decanting and add 500 ml of cold PBS to the cell pellet. Mix gently. Add 500 ml of cold, buffered 2% formaldehyde solution and mix again. Incubate at 2-8oC for 1h. Permeabilize cells with ethanol 4. Spin cells down by centrifugation for 5 min at 300 x g at 2-8oC, remove supernatant by aspiration or rapid decanting, wash once with cold 1 X PBS, then add 1 ml of 70% ethanol at – 20 oC drop-wise to the cell ...

ACCURATE MEASUREMENTS OF FLUORESCENCE INTENSITY SHIFTS CAN BE MADE BY CALIBRATING THE CYTOMETER WITH CHICKEN RED BLOOD CELLS¹ BACKGROUND Flow cytometers must be calibrated prior to fluorescence intensity measurements because of inherent instrument variability. To correct for this variability, a standard particle (fixed chicken red blood cells, or CRBCs) must be analyzed on the instrument prior to each experiment and photomultiplier tube (PMT) voltages adjusted accordingly to place the CRBC fluorescence emission peaks into predetermined target channels. MATERIALS 1. 10% Bleach solution 2. Double distilled water 3. Unstained cells of the type you are going to analyze in your experiment (negative control sample) 4. Cells stained with monoclonal antibodies of interest (positive control sample) 5. Glutaraldehyde-fixed CRBCs (Biosure, Cat#1004, Riese Enterprise. San Jose, CA) 6. 1x PBS + 2% newborn calf serum + 0.1% Sodium azide (PBSAz) METHODS ESTABLISH PMT VOLTAGE SETTINGS FOR ...

MATERIALS: 1. 1 X PBS (PBS-BSA, without sodium azide + 2% bovine serum albumin) 2. Human AB serum, heat inactivated (HAB, e.g., Irvine Scientific, CA) 3. Hoechst 33342 (HO342, Molecular Probes, OR) 4. Propidium iodide (PI, e.g., Calbiochem, CA) 5. 7-Amino-actinomycin D (7-AAD, e.g., Calbiochem, CA) 6. 37°C waterbath, centrifuge 7. Bucket with ice Preparation of stock solutions of HO342, PI, and 7-AAD HO342 Dissolve HO342 powder at a concentration of 1mg/ml in distilled H2O. Keep solution at 4°C protected from light. Solution can be stored for at least up to 6 months. PI Dissolve PI powder at a concentration of 1mg/ml in 1 X PBS. Keep solution at 4°C protected from light. Solution can be stored for at least up to 6 months. 7-AAD Dissolve 7-AAD powder (1mg) first in 50 microliters of absolute methanol, then add 950 microliters of 1 X PBS. Final concentration is 1mg/ml. Keep solution at 4°C protected from light. Solution can be stored for at least up to 6 mont...

I. SAMPLE (one or more of the following preparations) A. Suspensions of single cells from tissues B. Tissue culture cells C. Ficoll-hypaque separated mononuclear cells II. MATERIALS A. Antibodies 1. Primary antibodies: directly labeled with a fluorochrome. 2. Secondary antibodies: fluorochrome-labeled Streptavidin or antisera. B. 1 X PBS + 2% newborn calf serum + 0.1% sodium azide (buffer) C. Refrigerated centrifuge. OPTIONAL: A. Ammonium chloride lysing solution (see attached preparation method and procedure) B. RPMI 1640 media containing 10% or 20% serum Staining procedure 1. Place up to 30 X 106 Ficoll-hypaque separated cells in a 12 X 75 mm polypropylene sample tube and wash them once with buffer. 2. Add 0.5 ml of buffer to the pellet; then add the appropriate amounts of the monoclonal antibodies, e.g., FITC, PE, biotinylated or unconjugated antibodies. Usually 1/5 to 1/4 of the amount recommended by the manufacturer per cell number is sufficient for...

Q 1: When should I use fluorescence activated cell sorting over bulk separation methods like panning or magnetic bead separations? A: a) When very high purity (95%-100%) of the target population is required. b) For separation of populations that have a low density of receptors on their surface. c) For enrichment of populations on the basis of surface receptor density. d) For separations on the basis of multicolor staining. e) For separations on the basis of internal staining e.g of DNA or of internal antigens. Q 2: Will my cells be harmed by the sorting process? A: Generally, the cells will be not harmed through the process itself as long as they are maintained continually at a temperature, pH, and in media that is most suited to them. Q 3: How many cells do I need to prepare to recover 1 X 106 of a population that comprises 10% of the cells? A: 1 X 106 = 10 % target population x 50% recovery x 20 X 106 starting cell number. 50% recovery is a reasonable number, but th...

MATERIALS: 1. 2% formaldehyde solution (preparation method attached) 2. 1 X PBS (without sodium azide and serum) 3. 1 X PBS + 2% newborn calf serum + sodium azide (buffer) 4. Polyoxyethylensorbitan monolaureate (Tween 20) 5. Human AB serum (HAB, heat-inactivated) 6. Propidium iodide (PI)(e.g., from Calbiochem) 7. Ribonuclease A (RNase)(e.g., from Sigma) 8. 7-amino-Actinomycin-D (7-AAD, e.g., from Calbiochem) 9. 37°C water bath, refrigerated centrifuge. METHOD: Staining of surface antigens 1 x 106 PBS-washed cells from a single cell suspension are pelleted in a 12 X 75 mm culture tube. Then the staining proceeds as described in A1-3 for FITC-labelled antibodies and in B1-3 for simultaneous staining with FITC and PE-labelled antibodies. Fixation The pellet is resuspended in 0.875 ml of cold PBS and the suspension is mixed gently. Then, 0.125 ml of cold 2% formaldehyde solution is added and the mixture is immediately vortexed briefly. The suspension is incubated for at le...

MATERIALS: 1. 2% formaldehyde solution (preparation method attached) 2. 1 X PBS (without sodium azide and serum) 3. 1 X PBS + 2% newborn calf serum + sodium azide (buffer) 4. Polyoxyethylensorbitan monolaureate (Tween 20) 5. Human AB serum, heat-inactivated (HAB) 6. 37°C water bath, refrigerated centrifuge. METHOD: Fixation 1 x 106 PBS-washed cells from a single cell suspension are pelleted in a 12 X 75 mm culture tube. The pellet is resuspended in 0.875 ml of cold PBS and the suspension is mixed gently. Then, 0.125 ml of cold 2% formaldehyde solution is added and the mixture is immediately vortexed briefly. The suspension is incubated for at least 30 min or for up to 1 h at 4°C, centrifuged for 5 min at 250g, then the supernatant is removed. Permeabilization The pellet is gently resuspended in 1 ml of room temperature Tween 20 solution (0.2% in PBS) and the mixture is incubated for 15 min in a 37°C water bath. One ml of buffer is added and the suspension is sp...

I. SAMPLE - one or more of the following preparations A. Suspension of single cells from tissue (e.g., lymph node, spleen, bone marrow, placental cells) B. Tissue culture cells C. Ficoll-hypaque separated mononuclear cells II. REAGENT - hypotonic staining buffer for DNA Sodium citrate 0.25g Triton–x 100 0.75ml Propidium iodide 0.025g Ribonuclease A 0.005g Distilled water 250 ml We have kept this solution in a tightly-sealed bottle protected from light for several months without apparent loss of staining activity. III. STAINING 1. Place 1x106 cells into each tube. 2. Spin down samples and remove supernatant as completely as possible without disturbing the pellet. 3. Add 1 ml of the hypotonic DNA staining buffer to the pellet and mix well. 4. Keep samples at 4°C protected from light for 30 min or for a maximum of 1 h before acquisition on the flow cytometer. Note: Prolonged exposure to the hypotonic buffer can lead to an increase of debris in the sample...

7-Amino-actinomycin D (7-AAD) intercalates into double-stranded nucleic acids. It is excluded by viable cells but can penetrate cell membranes of dying or dead cells. I. MATERIALS: A. 7-Amino-actinomycin D (e.g., Cat #129935, Calbiochem, San Diego, CA) B. 1 X PBS with Ca2+ and Mg2+ C. Buffer: PBS (Ca2+ and Mg2+ free) +2% newborn calf serum (or 0.2% BSA) +0.1% sodium azide 7-AAD stock buffer: For long-term storage, store unopened vials of 7-AAD in the freezer. Dissolve 1 mg of 7-AAD powder by adding 50 microliters of absolute methanol directly to the vial. Mix well and add 950 microliters of 1 X PBS with Ca2+ and Mg2+ to achieve a concentration of 1 mg/ml. Store solution tightly closed and protected from light at 4°C. We have kept this solution for several months and have not observed loss in staining activity. II. METHOD: Stain your cells as outlined in the protocol for single color or dual-color staining with FITC and/or PE-labeled monoclonal antibodies. After ...

Propidium iodide (PI) intercalates into double-stranded nucleic acids. It is excluded by viable cells but can penetrate cell membranes of dying or dead cells. I. MATERIALS: 1. Propidium iodide (e.g., Cat #537059, Calbiochem, San Diego, CA) 2. Buffer: 1 X PBS (Ca2+ and Mg2+ free, e.g., Cat #9240, Irvine Scientific, Santa Ana, CA) +2% newborn calf serum (or 0.2% BSA) +0.1% sodium azide A) PI buffer: Dissolve PI in buffer at a concentration of 1 microgram/ml. Keep the solution tightly closed at 4°C protected from light. Discard after 1 month. B) PI stock buffer: Dissolve PI in buffer at a concentration of 500 micrograms/ml. Keep the solution tightly closed at 4°C protected from light. We have kept this solution for several months and did not observe loss in staining activity. II. METHOD: Stain your cells as outlined in the protocol for single-color staining with FITC-labeled monoclonal antibodies. A) After the last washing step resuspend your cell pellet in the PI...

METHOD 1: Formaldehyde preservative – 2% formaldehyde solution in protein-free phosphate-buffered saline (PBS). Prepare as follows: Add 2 g paraformaldehyde powder (e.g., Sigma, St. Louis, MO) to 100 ml of 1 X PBS. Heat to 70°C (do not exceed this temperature) in a fume hood until the paraformaldehyde goes into solution (note that this happens quickly as soon as the suspension reaches 70°C). Allow the solution to cool to room temperature. Adjust to pH 7.4 using 0.1 M NaOH or 0.1 M HCl, if needed. Filter and store at 4°C protected from light. METHOD 2: Formaldehyde preservative – 2% formaldehyde solution in protein-free PBS. Prepare as follows: 10% formaldehyde* 20 ml 10 x PBS 10 ml Distilled water 70 ml * 10% formaldehyde solution (e.g., Polysciences, Warrington, PA, ultrapure, Cat.#04018), depolymerized paraformaldehyde, EM grade, methanol-free solution.

I. SAMPLE A) Blood drawn into EDTA vacutainer tubes, keep at room temperature and process within 24 hours. B) Blood drawn into Heparin vacutainer tubes, keep at room temperature and process within 24 hours. II. REAGENTS A) Antibodies 1. Primary antibodies: usually purchased or your own monoclonals a) If these are conjugated with a fluorochrome (i.e., FITC, PE, or other) use the DIRECT STAINING PROCEDURE. b) If the primary antibody is not fluorescence-conjugated, use the INDIRECT STAINING PROCEDURE. 2. Secondary antibodies: fluoresceinated polyclonal antibody B) Buffer: PBS (e.g., Ca 2+ and Mg2+ free, Irvine Scientific, cat. #9240) + 2% newborn calf serum (or 0.2% BSA) + 0.1% sodium azide. C) NH4Cl Lysing Solution (see attached recipe) III. PROCEDURE DIRECT STAINING 1. First, add 50 microliters of BUFFER, then the appropriate amount of monoclonal antibody to the bottom of tubes. Note: Fo...

I.  SAMPLE – one or more of the following preparations A.  Suspension of single cells from tissue (e.g., lymph node, spleen, bone marrow, placental cells) B.  Tissue culture cells C.  Ficoll – hypaque separated mononuclear cells II.  REAGENTS A.  Antibodies 1.  Primary antibodies: usually purchased or your own monoclonals a.  if these are conjugated with a fluorochrome (e.g., FITC or PE) use the DIRECT STAINING PROCEDURE b.  if the primary antibody is not conjugated to a fluorochrome, use the INDIRECT STAINING PROCEDURE 2.  Secondary antibodies: fluoresceinated polyclonal antibodies B.  BUFFER: Phosphate-buffered saline (PBS Ca 2+ and Mg 2+ free) + 2 % newborn calf serum (or 0.2% BSA) + 0.1% sodium azide. C.  Formaldehyde preservative: 2% solution in protein-free PBS D.  PBS: protein and azide free Buffer and formaldehyde s...

If your cells have many Fc-receptors on the cell surface (in particular monocytes, macrophages) or they have been cultured in serum free medium, it is advisable to block nonspecific binding of monoclonal antibodies by pre-incubation of cells with human AB serum (HAB). Note that for staining of whole blood this is not necessary, because serum in high concentration is present during staining. I. REAGENTS: A. Antibodies 1. Primary antibodies: usually purchased or your own monoclonals a. If these are conjugated with a fluorochrome (i.e., FITC, PE, or other) use the DIRECT STAINING procedure. b) If the primary antibody is not conjugated to a fluorochrome, use the INDIRECT STAINING procedure. 2. Secondary antibodies: fluorochrome-conjugated polyclonal antibody B. Buffer: PBS (Ca2+and Mg2+ free, e.g., Cat. #9240, Irvine Scientific, Santa Ana, CA) + 2% newborn calf serum (or 0.2% BSA) + 0.1% sodium azide. C. HAB...

I. Dilute the fluorochrome-conjugated antibody further. If it is too concentrated, background may increase due to an increase in non-specific interactions. II. BEFORE applying the primary antibody, incubate with excess protein such a bovine serum albumin (BSA), non-fat dry milk, or normal serum from the same host as the labeled secondary antibody. This step may reduce background by blocking non-specific interactions between the primary antibody and the cell surface or intracellular structures. III. AFTER applying the primary antibody, incubate with 5% to 10% normal serum from the same host as the labeled secondary antibody. This step may reduce unwanted interactions between the secondary antibody and the primary antibody, the cell surface, or intracellular structures. It is possible to avoid this step by diluting the labeled antibody with normal serum from the same species. This works well in many cases, but occasionally it can lead to the formation of immune complexes between...