FACS staining of human breast cancer cores for ALDH, CD24, CD44, EpCAM, and Lineage negative cells

Transcription

1 .0 Purpose The purpose of SOP is to describe FAC staining of human breast primary tissue for ALDH, and lineage. 2.0 Scope SOP is intended to cover all resources, personnel and equipment in the BCR laboratory. 3.0 Stains Channel Marer MAb Clone Company Catalog Lot Conc Stain vol (X) FITC Aldefluor NA NA VMCC 0.5 ul PE EpCAM Mouse EBA- BD ul PE-Cy7 CD24 IgG2a ML5 Biolegend 320 B ul APC CD44 Mouse IgG2b G44-26 BD ul Alexa Live-Dead NA NA Invitrogen L NA ul Alexa Cells Streptavidin (SAv) NA NA Invitrogen S mg / ml Total ul 76.5 ul 00 ul Channel Marer MAb Clone Company Catalog Lot Conc Stain vol (X) FITC Aldefluor NA NA VMCC 0.5 ul PE Isotype Mouse X40 BD ul PE-Cy7 Isotype Mouse X40 BD ul APC Isotype Mouse X40 BD ul Alexa Live-Dead Invitrogen L NA ul Alexa Cells Streptavidin (SAv) NA NA Invitrogen S mg / ml Total ul 92.5 ul 00 ul NOTE: Biotin conjugated antibodies are used first, then stain with streptavidin and other antibodies Page

2 Biotinylated antibodies: For Stain and Isotype CORE tubes Channel Marer Lineage MAb Clone Company Catalog Lot Conc Vol (ul) Biotin CD45 All except RBC & platelets, HI30 ebioscience E HLA- DR B, mono, macro, DC, active T CD4 Mono / macro IgG2b,, CD3 Endo, CD4 Platelets / Mega CD9 B-cell, CD235a RBC IgG2b, CD56 NK, CD3 T-cell, CD6 Macro, NK, neutro CD40b Fibroblasts, LN3 ebioscience D3 ebioscience WM59 ebioscience E E E MEM-06 Abcam AB HIB9 ebioscience E HIR2 ebioscience CMSSB ebioscience UCHT ebioscience E E E ebiocb6 ebioscience E A2 Biolegend B Materials/Reagents Needed ml FACS tubes (2 x 75mm) with BLUE 35uM filter cap (BD ) ml FACS tubes (2 x 75mm) (BD ) 4.3. ul microcentrifuge tubes [sterile] 4.4. Aldefluor reagents [aliquot] a. One aliquot of Aldefluor substrate [aa BAAA] Page 2

3 b. DEAB [blac cap glass vial,.5mm in ethanol] c. Aldefluor Assay buffer 4.5. Live-Dead Near IR (aa Alexa /APC-Cy7 channel) [Invitrogen L09] a. Already diluted in 00% DMSO; may be frozen; 4.6. BD CompBead Plus Anti-Mouse Ig and Negative control set [BD ] a. Anti-Mouse Ig, [Component ; BLUE cap dropper] b. Negative control (BSA) [Component ; WHITE cap dropper] 4.7. ArC Bead it [Invitrogen A0346] a. Reactive Beads [Component A GREEN cap] b. Negative Beads [Component B white cap] 4.8. Antibodies. See tables for catalogs 4.9. PBS (or HBSS) with 3% FBS, sterile filtered 4.0. MCF7 (or T47D or SUM59) cells actively growing; at least 0.5x0^6 cells 4.. Digested core biopsy (aa CORE) tumor cells 4.2. Cytometer with 488nm and 633nm lasers (56nm laser is ideal for ALDH/PE compensation) 5.0 Procedure (for core sample and MCF7) 5. Count cells in CORE sample a. Centrifuge CORE sample for 5min at 4C at g b. Aspirate supernatant c. Add 300 ul Aldefluor staining buffer to pellet and measure volume (usually uL) d. Add 0uL cells to 30uL trypan blue and count on hematocytometer 5.2. Label 6 regular FACS tubes for compensation: Unstained, ALDH, PE, PE-Cy7, APC, Ax 5.3. Label 6 regular FACS tubes for each sample: Stain, DEAB, and Isotype a. Label one set CORE and the other set MCF Label 2 blue-cap filter tubes CORE and MCF Label microcentrifuge tubes: DEAB, Stain, Isotype, Biotin 5.6. Filter CORE and MCF7 cells into separate blue-cap FACS tube: a. Pre-wet filter cap tubes with 0.5mL PBS/FBS b. Add all cells to filter c. Add ul Aldefluor staining buffer to filter to exchange buffer d. Centrifuge tubes for 5min at 4C at g e. Aspirate supernatant, taing care to minimize disruption of cell pellet f. Resuspend MCF7 cells with 350 ul Aldefluor staining buffer; store at RT g. Resuspend CORE cells with 30 ul Aldefluor staining buffer i. Measure volume of cells ii. If needed, adjust volume with Aldefluor staining buffer to 00 ul 5.7. Stain CORE cells with biotin lineage coctail a. Mae master mix in ul microcentrifuge tube i. 0.5jL CD4 and ul all other antibodies (0.5 ul total) b. Aliquot CORE cells to regular FACS tube i. 75 ul cells [from Step 5g] to Stain tube. Add 20 ul Aldefluor staining buffer to Stain tube (95 ul total) ii. 25 ul cells [from step 5g] to Isotype tube. Add 70 ul Aldefluor staining buffer to Isotype tube (95 ul total) c. Add 5.25 ul of biotin lineage coctail to Stain and Isotype tubes Page 3

4 d. Incubate for 30 min at RT 5.8. Dilute DEAB a. Add 90 ul Aldefluor staining buffer to DEAB microcentrifuge tube b. Add 0 ul DEAB stoc [blac cap glass vial] into staining buffer 5.9 Add compensation beads to tubes: a. PE, PE-Cy7, and APC tubes receive: i. One drop BD CompBead Plus Anti-Mouse Ig beads [BLUE cap dropper] ii. One drop BD CompBead Plus Negative control (BSA) [white cap dropper] b. Ax tube receives drop ArC Reactive beads [Component A GREEN cap] 5.0 Add antibodies to beads in appropriate compensation tube a. 3 ul EpCAM PE to PE tube b. ul CD24 PE-Cy7 to PE-Cy7 tube c. 3 ul CD44 APC to APC tube 5. Add ul Live-Dead stoc to Ax tube 5.2 Add 00uL MCF7 cells to unstained and ALDH tubes [from Step 5f] a. Add 0.5 ul Aldefluor reagent to ALDH tube 5.3 After ~30min, add ul Aldefluor staining buffer to CORE Stain and Isotype tubes 5.4 Centrifuge both CORE tubes for 5 min at 4C at g 5.5 Aspirate supernatant, taing care to minimize disruption of cell pellet 5.6 Resuspend CORE Stain and Isotype cells with 30 ul Aldefluor staining buffer a. Measure volume of cells b. If needed, adjust volume with Aldefluor staining buffer to: i. Stain 76.5 ul ii. Isotype 92.5 ul 5.7 Mae Stain and Isotype master mixes in ul microcentrifuge tubes: a. For CORE and MCF7, mae 3X Stain mix Channel Marer Stain (X) Stain (3X) Stain (4X) FITC Aldefluor 0.5 ul.5 ul 2 ul PE EpCAM 0 ul 30 ul 40 ul PE-Cy7 CD24 ul 3 ul 4 ul APC CD44 0 ul 30 ul 40 ul Alexa Live-Dead ul 3 ul 4 ul Alexa SAv ul 3 ul 4 ul Total volume 23.5 ul 70.5 ul 94 ul Vol/tube 23.5 ul 23.5 ul 23.5 ul b. For CORE and MCF7, mae 3X Isotype mix Channel Marer Stain (X) Stain (3X) Stain (4X) FITC Aldefluor 0.5 ul.5 ul 2 ul PE Iso 2 ul 6 ul 8 ul PE-Cy7 Iso 2 ul 6 ul 8 ul APC Iso ul 3 ul 4 ul Alexa Live-Dead ul 3 ul 4 ul Alexa SAv ul 3 ul 4 ul Total volume 7.5 ul 22.5 ul 30 ul Page 4

5 Astrios Vol/tube 7.5 ul 7.5 ul 7.5 ul 5.8 Aliquot MCF7 cells to regular FACS tubes a ul cells [from Step 5f] to Stain tube b. 25 ul cells [from Step 5f] to Isotype tube i. Add 67.5 ul Aldefluor staining buffer to Isotype tube 5.9 Aliquot 5 ul diluted DEAB [from Step 7] to DEAB FACS tubes; recap tubes 5.20 Add 7.5 ul Isotype master mix to Isotype FACS tubes 5.2 Add Stain master mix to Stain FACS tube a. Add 23.5 ul master mix to cells b. Immediately transfer 50 ul of these cells to DEAB tube with the 5 ul diluted DEAB. Verify that cells and DEAB are mixed c. Proceed to next sample and repeat Steps 6a and 6b 5.22 Incubate all tubes in incubator at 37C for 30 min to hour; cover with foil if paranoid or incubator is constantly being opened 5.23 Every 5 minutes, remove tubes from incubator and quicly shae tube rac to resuspend cells 5.24 Add ul Aldefluor staining buffer to Stain, DEAB, Isotype tubes 5.25 Add ul PBS / 3%FBS to Unstained, ALDH, PE, PE-Cy7, APC, Ax compensation tubes 5.26 Centrifuge all tubes for 5 min at 4C at g 5.27 Aspirate supernatant from all tubes a. Compensation tubes (except Ax) should have very small visible pellet b. Ax tube will have diffuse pellet 5.28 Add ul Aldefluor staining buffer to Stain, DEAB, and Isotype tubes and resuspend pellet Add ul PBS / 3%FBS to Unstained, ALDH, PE, PE-Cy7, and APC compensation tubes 5.30 Add ul PBS / 3%FBS to Ax compensation tube a. Add drop of ArC NEGATIVE beads [Component B white cap] 5.3 Place tubes on ice 5.32 Aliquot 000 ul PBS / 3%FBS to 3 regular FACS tubes for collecting stem cell (and bul) fractions a. ALDH+EpCAM+ b. CD24-CD44+EpCAM+ALDHc. Bul 5.33 Use MCF7 cells to setup cytometer voltages 5.34 Collect viable from CORE Isotype and DEAB for bul tumor cells for comparison Fraction Default gates Isotype DEAB Stain Bul ALDH+EpCAM+ CD24-CD44+EpCAM+ALDH- R+R2+R3+R4 R+R2+R3+R4+R5+R20 R+R2+R3+R4+R5+R4 Page 5

6 MoFlo Fraction Default gates Isotype DEAB Stain Bul ALDH+EpCAM+ CD24-CD44+EpCAM+ALDH- R+R2+R3+R4 R+R2+R3+R4+R4+R3 R+R2+R3+R4+R5+R6 6 Applicable References 7 Change Description Revision Date Reference Description of Change Page 6