Application Protocol: CD45 CK Immunostaining for patient blood
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1 REPRODUCTION AND USE This document is protected by copyright and it cannot be used or shared without permission from Vortex Biosciences, Inc. Such permission is given on condition that Vortex Biosciences is acknowledged whenever this protocol is reproduced or is published in whole or in part. PURPOSE This Application Protocol describes a method for the immunofluorescence staining of cells enriched from a human blood sample with Vortex device. This Protocol is using an antibody cocktail against human Cytokeratin marker (subtypes CK1 to CK8, CK10, CK14-16 and CK19) to identify epithelial cancer cells, as well as CD45 marker to identify white blood cells and DAPI to stain cell nucleus. This immunostaining requires the fixation and permeabilization of the collected cells. Figure 1: Simplified schematic of antibody binding in different types of cells MATERIALS Consumables Normal waste container. PFA waste container. Paper towels. Pipette tips (p10, p200, p1000). Tin Foil and Parafilm. Page 1 of 7
2 15 and 50 ml Falcon tubes. 5, 10 and 25 ml serological pipettes. Eppendorf tubes. Luer Lock BD syringes. Sterile syringe filters (Corning 28mm Diameter Syringe Filters, 0.2µm Pore SFCA Membrane, # ). Equipment Fume hood. Centrifuge with well plate holders, Beckman GS-6R or equivalent. Tube rack. Pipetter (p10, p200, p1000). Pipet-aid. Vortexer. Timer. Relevant Personal Protective Equipment (PPE). Chemicals and Reagents (these should NOT be replaced without further validation) Cells collected in a sterile 96-well plate (CELLSTAR Greiner F-bottom with lid, # ). Sterile PBS (Gibco DPBS 1x # or Lonza BioWhittaker PBS # F (without calcium and magnesium) or equivalent. Prepare an aliquot under a fume hood on the same day. Deionized water (HyClone Hypure WFI quality Water, Thermo Fisher # SH or Invitrogen UltraPure Distilled Water, Invitrogen # ). 4% Paraformaldehyde (Electron Microscopy Sciences, 10 x 10 ml, Cat ) 10% Normal Goat Serum (100 ml, with 0.1% Sodium Azide, Invitrogen, # 50062Z). 10 mg DAPI, dilactate, (Life Technologies # D3571). 4% PFA fixed human white blood cells. 4% PFA fixed human cancer cells, such as MCF7 cancer cell lines. Triton-X100 (Sigma, #T ml). Antibodies: Markers Clone Fluorochromes Vendor Reference # Concentration (mg/ml) Mouse anti-cytokeratin CAM 5.2 FITC BD Biosciences Mouse anti-cytokeratin CK3-6H5 FITC MACS Miltenyi Unknown Mouse anti-pan-cytokeratin AE1/AE3 AF488 ebioscience Mouse anti-cd45 HI30 PE BD Pharmingen Unknown Do not freeze the antibodies or antibody cocktails. Do not expose them to direct light during storage or incubation. Do not replace these antibodies by other brands neither clones without further validation. Page 2 of 7
3 PROCEDURE Preparing Reagents, BEFORE day of staining 1) 4% PFA a) Under a fume hood aliquot 4% paraformaldehyde in 1.5 ml Eppendorf tubes. b) Write down the aliquot date and concentration. Store these aliquots at -20 C. 2) 0.4% Triton X-100 a) In a 50 ml Falcon tube, dilute 100 µl Triton-X100 in 25 ml sterile PBS 1X. b) Sterifilter (0.2 µm) to avoid the presence of any debris. Assume that 300 µl is lost through the filter. c) Aliquot by 5 ml in 15 ml Falcon tubes. d) Write down the aliquot date and concentration. Store these aliquots at RT for 1 year. 3) DAPI at 5mg/mL a) In the dark, re-suspend the initial vial at 5 mg/ml by adding 2 ml of deionized water. Handle with care, as DAPI is a known mutagen. b) Aliquot by 0.5 ml in 1.5 ml Eppendorf tubes. c) Write down the aliquot date and concentration. Store these aliquots at -20 C, protected from light. 4) DAPI at 1mg/mL a) In the dark, in a 15 ml Falcon tube, add 2.0 ml deionized water to 0.5 ml DAPI 5 mg/ml. b) Sterifilter (0.2 µm) to avoid the presence of any debris, which would fluoresce within the well during the imaging process. Handle with care, as DAPI is a known mutagen. c) Aliquot by 0.5 ml in 1.5 ml Eppendorf tubes. d) Write down the aliquot date and concentration. Store these aliquots at -20 C, protected from light. e) Once opened, use the aliquot within 1 month and store at 4 C. Write down the opening date. Preparing Reagents, DAY of staining 5) 10% Goat Serum a) Sterifilter (0.2 µm) 10% Goat Serum to avoid the presence of any debris. Assume that 300 µl is lost through the filter. b) Prepare this solution, considering that you need 10% Goat Serum for the Permeabilization step, the Blocking Step and the Antibody Staining Step (or about 500 µl per sample). 6) Permeabilization buffer a) Mix an equal volume of 0.4% Triton-X100 and 10% Goat Serum considering that you need 200 µl per sample. 7) Antibody Mix a) For each well of a 96-well plate, prepare the following mix in the dark, on the day of the experiment. 5 µl of CK CAM5.2-FITC (BD Biosciences), i.e. ratio of 1:40, Page 3 of 7
4 5 µl of Pan-CK-FITC (MACS Miltenyi), i.e. ratio of 1:40, 5 µl of CK AE1-AE3-AF488 (ebioscience), i.e. ratio of 1:40, 10 µl of CD45-PE (BD Biosciences), i.e. ratio of 1:20, 1 µl of DAPI at 1mg/mL, i.e. ratio of 1:200, 174 µl of 10% Goat Serum. b) Multiply these volumes by the number of wells that need to be stained (processed wells and 2 control wells). Add equivalent of 2 other wells as a safety volume. c) Mix by pipetting. Store this cocktail at 4 C in the dark until ready to use. Do not store the cocktail for longer than 6 hours. Fixation, Permeabilization and Staining in 96 well-plate 8) Preliminary Wash a) Fill the wells to be stained with 1X PBS. b) Centrifuge 1. (up to 800 RCF, low brake, at RT). Note: unless specified, all the centrifugation steps will be performed with these settings. c) Aspirate 200 µl from the well using a P200, using the sides of well to aspirate, without touching the bottom of the well. Leave a liquid volume of about 150 µl. 9) Fixation If the cells were already fixed, proceed directly to Step 10. a) Under the fume hood add 4% PFA, 200 µl volume per well. Incubate for 10 min, at RT. b) Centrifuge 2. Aspirate 200 µl with a P200. Add 200 µl 1X PBS. Collect the PFA waste in a specific PFA waste container. c) Centrifuge 3. Aspirate 200 µl with a P200. Add 200 µl 1X PBS. d) Centrifuge 4. Aspirate 200 µl with a P200. Add 200 µl 1X PBS. e) Centrifuge 5. Aspirate 200 µl with a P200. If necessary, the experiment can be stopped at this point. Add 1X PBS to the wells, wrap the plate with parafilm and tin foil, label the plate with sample ID + staining date + steps performed + user initials and store it at 4 C. Do not store a plate longer than 6 months. If not stopping, go directly to Step ) Control Wells a) Using a P200, add more than 500 fixed white blood cells in a separate well as a positive control for CD45-PE staining. b) Using a P200, add more than 500 fixed MCF-7 breast cancer cells or equivalent in a separate well as a positive control for CK-FITC staining. c) Fill the control wells with 1X PBS. d) Centrifuge 6. Aspirate 200 µl with a P200. e) Check the control wells under the microscope, to make sure there are enough cells for a relevant control before moving to the Permeabilization step. Page 4 of 7
5 11) Permeabilization a) Add 200 µl of Permeabilization buffer per well. Incubate for 7 min, at room temperature. b) Centrifuge 7. Aspirate 200 µl with a P200. Add 200 µl 1X PBS. c) Centrifuge 8. Aspirate 200 µl with a P200. Add 200 µl 1X PBS. d) Centrifuge 9. Aspirate 200 µl with a P200. Add 200 µl 1X PBS. e) Centrifuge 10. Aspirate 200 µl with a P ) Blocking a) Add 200 µl of 10% Goat Serum per well. Incubate for 30 min, at room temperature. b) Centrifuge 11. Aspirate 200 µl with a P ) Antibody Staining a) In the dark, add 200 µl of Antibody Mix. Wrap the plate in tin foil. Incubate for 1 hour at room temperature in the dark. b) Centrifuge 12. Aspirate 200 µl with a P200. Add 200 µl 1X PBS. c) Centrifuge 13. Aspirate 200 µl with a P200. Add 200 µl 1X PBS. d) Centrifuge 14. Aspirate 200 µl with a P200. Add 200 µl 1X PBS. e) Centrifuge 15. Aspirate 150 µl with a P ) Well-plate Labeling and Storage a) Place some parafilm over the wells that were stained. Wrap the well plate with tin foil to protect them from the light. b) Label both the plate and the foil with: sample ID + staining date + steps performed + user initials. c) Store the well plate at 4 C for long-term storage in designated space. Clean up Bring all equipment and supplies back. Spray & wipe down all surfaces with 70% ethanol. Keep the shared space clean for next users. IMAGING 1. Refer to the table below to find the best channels to image the different fluorochromes. Antibody Clone Fluorochrome Mouse anti-cytokeratin CAM 5.2 FITC Mouse anti-cytokeratin CK3-6H5 FITC Mouse anti-pan-cytokeratin AE1/AE3 AF488 Mouse anti-cd45 HI30 PE Page 5 of 7
6 2. Picture samples Figure 2: Staining of white blood cells (WBC) and MCF7 (epithelial cancer cells). Scale bar: 20 µm TROUBLESHOOTING Problem Cause Solution The cells in the sample wells are not stained. The cells may not express the antigens, or were damaged. Repeat the staining by selecting specific antibodies (see more application protocols from Vortex Biosciences). Verify that the cells were stored at 4 C, in the dark, for less than a year. The cells in the control wells are not stained. The cells are damaged or old. Verify that the cells were stored at 4 C, in the dark, for less than a year. The cells, in BOTH the sample wells and the control wells are not stained. The staining went wrong. By user mistake or maybe the antibodies or DAPI are contaminated or expired. Repeat the staining but with controls only, to confirm that all antibodies or DAPI were added. If controls still don t stain (or stain very faintly), change the Ab or DAPI stock. Be sure to perform the staining step in the dark, including Ab preparation / dilution. There is debris in the wells, making cell observation difficult. PBS or another reagent may have been contaminated. Make sure you use fresh, sterile PBS. Make sure you sterifilter reagents throughout the protocol. Page 6 of 7
7 IMPORTANT NOTES 1. Wear personal protective equipment. Always wear goggles, lab coat, and gloves. 2. Stain in the dark. While working with the fluorescently-labeled antibodies, always make sure you are in the dark to avoid fluorophore bleaching. During incubation times, keep the well plate under tin foil. 3. Keep track: Write down the date, sample number, type of sample, the procedures, and your initials. Write this information on the well plate, AND the foil around the well plate. Mark clearly which wells correspond to which sample. 4. Avoid cross-contamination between wells. Do not use the same pipette tips between the different wells, especially if the cells collected in these wells are from different patients. Do not use the same pipette tips when removing the PBS from multiple wells during the wash steps. You could accidentally move the cells from one well to another. Do not collect 2 different samples in wells close to each other, as some sample may splash over the next well when there are full of liquid. Ideally, you would collect samples in the top left, top right, bottom left, and bottom right corners of the well plate. Control cells could be located in the center of the well plate. Cover the wells with parafilm before long-term storage to avoid evaporation. Page 7 of 7
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