Lab Protocols

Abstract In an earlier article from this laboratory, the current methods developed to detect apoptosis in cells and tissues were highlighted, along with the challenges in their interpretation. Recent discoveries concerning the underlying biochemical mechanisms of apoptotic effector pathways have made possible further assays that allow a more direct measure of the activation of the apoptotic machinery in cells. This article summarizes some of these newer methods and extends the interpretation of the more classical assays of apoptosis in a defined cell system. We present data in KB and PC3 cell model culture systems induced to undergo apoptosis by the plant toxin ricin. Using a modified in situ nick translation assay (ISNT) with either Bodipy or BUdR labeling, we confirm that most cells showing altered nuclear morphology do not show reactivity with thi...

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...