Lab Protocols

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...