HUMAN EMBRYONIC STEM CELL (hesc) ASSESSMENT BY FLOW CYTOMETRY USING DIRECTLY CONJUGATED ANTIBODIES

Transcription

1 HUMAN EMBRYONIC STEM CELL (hesc) ASSESSMENT BY FLOW CYTOMETRY USING DIRECTLY CONJUGATED ANTIBODIES OBJECTIVE: The pluripotency quality of human Embryonic Stem Cells (hescs) can be assessed by using flow cytometry to measure percentage of cells in all population expressing hesc-specific pluripotency markers in the cell population. This Standard Operating Procedure (SOP) describes how to stain specific cell surface markers by fluorochrome-conjugated antibodies, followed by analysis using flow cytometry and data reporting. This SOP applies to hescs grown in the presence of inactivated Mouse Embryonic Fibroblasts (MEFs). SCOPE: This procedure applies to all Massachusetts laboratory personnel responsible for characterization of hescs by flow cytometry. RESPONSIBILITY: It is the responsibility of the Laboratory Operations Manager and Quality Assurance Officer to ensure all required personnel are properly trained in and follow this SOP. SAFETY: All laboratory personnel should be in compliance with UMASS Employee Health and Safety regulations when working in the laboratory. Specifically, wear protective equipment (lab coat, disposable gloves) when working in the lab. ABBREVIATIONS AND DEFINITIONS hescs: Human Embryonic Stem Cells MEF: Mouse Embryonic Fibroblast WB: Wash Buffer FB: Fixation Buffer SOP: Standard Operating Procedure UMASS: University of Massachusetts Medical School HI-FBS: Heat-Inactivated Fetal Bovine Serum REFERENCES Not applicable 1. MATERIALS REQUIRED 1.1. EQUIPMENT Flow cytometer equipped with 488 nm, 405 nm, and 633 nm lasers Sterile biosafety cabinet (tissue culture hood) Beckman tabletop centrifuge Refrigerator Hemocytometer or other cell counter Inverted Microscope Vortex Micropipettors: 92

2 P20 ( µl) P200 ( µl) 1.2. SUPPLIES 40 micron cell strainer (BD Falcon ) 12 x 75 mm round bottom 5 ml tubes (BD Falcon ) Tips to fit P20 µl and (Corning 4821 and 4823) P020 µl pipettors 5 ml serological pipettes (Costar 4487) 10 ml serological pipettes (Costar 4488) Pasteur pipettes (Fisher brand D) 15 ml centrifuge tubes (BD Falcon ) 50 ml centrifuge tubes (BD Falcon ) Black extra-fine alcohol proof pen (VWR ) Disposable nitrile gloves (World Wide Medical Supplies ) 70% ethanol (Fisher NC ) 1.3. REAGENTS PBS (Invitrogen ) (Dulbecco s Phosphate-Buffered Saline, Ca/Mg-free) Heat-Inactivated Fetal Bovine Serum (HI-FBS) (Invitrogen ) 1 x Trypsin-EDTA (0.05%) (Invitrogen 25300) MEF culture medium (see SOP-RP-002 Preparation of Mouse Embryonic Fibroblast (MEF)) 16% methanol-free Formaldehyde stock solution (Polysciences Inc, ) Antibodies : Antibody Fluorochrome Vendor Catalog Number CD29 ALEXA 488 Serotec MCA2298A488 Isotype control ALEXA 488 BD Pharmingen SSEA4 PE R&D Systems FAB1435A Isotype control PE R&D systems IC007P SSEA1 ALEXA 647 Biolegend PROCEDURE Isotype control ALEXA 647 Biolegend REAGENT PREPARATION WASH BUFFER (WB): 1. Prepare 100 ml WB according to the volumes listed in the table below. Adjust the volumes of reagents if another quantity of wash buffer is to be prepared. 1 x PBS Fetal Calf Serum 98 ml 2 ml 2. Store the WB at 4 C. It is stable up to one month at 4 C. 93

3 FIXATION BUFFER (FB): 1. Add 2 ml of methanol-free formaldehyde (16%) to 14 ml of PBS (final 2% formaldehyde solution). 2. Make fresh and keep on ice. 3. Discard at the end of the day DETACH AND COUNT CELLS IN A CELL CULTURE LAB Note 1: The steps below apply to a 6-well culture plate. If cells are cultured in other culture vessels, adjust the reagent volumes proportionately. Note 2: 1-2 x 10 6 hescs can be harvested from one well of a 6-well plate when colony confluence is between 60-70%. A minimum of 3 x 10 6 cells are required for this procedure. In a biosafety cabinet: 1. Aspirate spent medium from culture wells. 2. Rinse twice with 3 ml/well of 1 x PBS. 3. Aspirate after each rinse. 4. To detach cells, add 1 ml of 1 x Trypsin-EDTA to each well. 5. Incubate at 37ºC for 2-4 minutes. Using a microscope, confirm that cells have detached from plate. 6. Using a glass pipette, gently scrape cells and pipette up and down to break up the cell clumps. 7. Within 5-6 minutes after adding the Trypsin-EDTA solution to inactivate Trypsin enzyme, add 2 ml MEF culture medium to each well. 8. Pool the detached cells into one 15 ml or 50 ml tube. 9. Centrifuge the tube for 5-7 minutes at 200 x g (1000 rpm). 10. Resuspend the cell pellet in 3 ml/well of 1x wash buffer. 11. Add the cells to the center of a 40 micron cell strainer. Allow the cells to pass through the filter by gravity. 12. Rinse the cell strainer top reservoir with 2 ml of 1x WB. 13. Count cells in a Hemocytometer or other cell counter. 14. Centrifuge the tube containing cells for 5-7 minutes at 200 x g (1000 rpm). 15. Resuspend the cell pellet at 3 x 10 6 cells/ml in 1 x wash buffer. 16. Continue with the staining procedure below STAIN AND FIX CELLS IN A MOLECULAR LAB 1. Label six FACS tubes (12 x 75 mm round bottom test tubes) as 1 to 6 sequentially. 2. To each tube except #1, add 100 µl of the cell suspension (about 3 x 10 5 cells/tube). 3. Add the rest of the cells (more than 100 µl ) to tube #1. 4. To each tube except #1, add 10 µl of each antibody according to the table below: 94

4 Tube ID Stained for Antibodies added 1 nothing no 2 Control Ig IgG-ALEXA488 + IgG-PE + IgM- 3 MEF, CD29 CD29-Alexa488+ IgG-PE + IgM- 4 hesc, SSEA4 IgG-ALEXA488 + SSEA4-PE + IgM- 5 Differentiating cells, SSEA1 IgG-ALEXA488 + IgG-PE + SSEA1-6 All three markers CD29-Alexa488+ SSEA4-PE + SSEA1-5. Incubate for 30 minutes at 4 C in dark by wrapping the tubes in foil. 6. Add 2 ml of 1 x Wash Buffer, centrifuge for 10 minutes at 1000 rpm. 7. Decant the supernatant and resuspend the cell pellet in 100 µl of 1 x FB. Vortex and incubate the cells at room temperature for 30 minutes in the dark. 8. Add 2 ml of 1 x PBS, centrifuge for 10 minutes at 1300 rpm. 9. Decant the supernatant, and resuspend the cells in 300 µl of 1 x Wash Buffer. 10. Submit the samples to UMASS Flow Cytometry Core Facility, University of Massachusetts Medical School, 5 th Floor, or store the samples at 4-8 C in refrigerator until submitted to the Core Facility. Samples should be analyzed done within one week DATA ACQUISITION AND ANALYSIS 1. Flow samples will be analyzed by UMASS Flow Cytometry Core Facility. 2. Data printouts should be available from the Core within three days of sample submission. 3. RESULTS REPORTING AND ACCEPTABLE RANGE 1. Copy a Flow Data Log Sheet. 2. Summarize the results in the table on the Flow Data Log Sheet. The format of the table is shown below: Marker CD29+SSEA4- SSEA1- CD29- SSEA4+SSEA1- CD29-SSEA4- SSEA1+ Target population Actual positive (%) MEF <20% hesc >90% Differentiating cells <10% Acceptance criteria (%) Pass or fail 3. Fill out the Flow Data Log Sheet completely and attach it together with data sheets (date and sign each sheet) from the Core and place in the appropriate binder or folder. 4. Notify appropriate staff about the test results and the decision made regarding the test sample. 95

5 FLOW DATA LOG SHEET Provider s cell ID Bank code Passage number Lot number Additional information about the test sample Staining done by date Analysis done by date Test results: Marker CD29+SSEA4- SSEA1- CD29- SSEA4+SSEA1- CD29-SSEA4- SSEA1+ Target population Actual positive (%) MEF <20% hesc >90% Differentiating cells <10% Acceptance criteria (%) Pass or fail Decisions made (circle one): If the results fail the acceptance criteria, either discard the lot of cells or use SOP-CC-006 Manual Passage of Human Embryonic Stem Cells (hescs) on Fresh Mouse Embryonic Fibroblast (MEF) Plates to manually select hescs for further culture. If the results pass the acceptance criteria, release the lot of cells. Recorded by Date (month/day/year) Verified by Date (month/day/year) Attach this sheet to an appropriate binder or folder when complete 96