Dynabead Sort (CD10 EpCAM) ( Click to Download )

  1. Prepare Dynabeads for sorting:
    1. Remove 400 ml beads, resuspend in 1 ml B2 to wash, magnet 1’, resuspend 400 ml B2.  Add 200 ml to one tube + 50 ml aCD10 (10 µg Santa Cruz Clone SS2/32); add 200 ml to one tube + 30 ml aESA (30 µg AbdSerotec VU-ID9).
  2. Incubate on tube rotator for 1+ hour
  3. Wash CD10 and EpCAM beads with 3 x 1 ml B2 and resuspend in 200 ml B2, keep on ice.
Fraction Estimated cells Final count cells/ml Total cells
(U) Unsorted
(M) CD10+
To sort (CD10-) NA NA
(L) EpCAM+CD10-
(D) ESA-CD10-

 

Experiments (depends on needs):

  1. Plating for adherent colony growth to generate vHMECs

250K per 10cm dish in MC from Unsorted, CD10+, EpCAM+, grow for 10-14 days before first passage. Plate 500K cells for next two passages and then plate 250K cells to wait for variants.

  1. Plating for 3D culture growth on collagen gels

10K per well x 3 wells plated in 4-well chamber slides

Collagen Mix: 1.5 mls for 3×4-well slides

150 ml 10x PBS

366 ml 4.1 mg/ml Rat Tail Collagen (Millipore)

6 ml 1N NaOH

978 ml sterile H20

Keep everything cold!  100 ml per gel, allow to set up at RT or 37C for 30

minutes at minimum!  Add cells in 1 ml of MC media + 2% matrigel per well. Re-feed every 3-4 days

 

  1. 4-well chamber slide for colony outgrowth for immunofluorescence (can coat wells with dilute collagen or matrigel if desired):
    1. 5000 cells per well
    2. 20K in 3.2 ml MC
    3. 800 µl per well x 4 wells
  2. Matrigel coated wells of a 8-well chamber slide for colony outgrowth in a matrigel overlay assay (pipet and spread 50 µl of matrigel per well, let solidify 15-30 min at 37C before plating cells).
    1. 5000 cells per well
    2. 20K in 800 µl MC
    3. dilute 1:2 in 800 µl MC + 4% MG
    4. 400 µl per well
    5. re-feed with MC + 2% MG every 3-4 days
  3. 6-well non-adherent plate for Mammosphere outgrowth:
    1. 20,000-40,000 cells per well (10K-20K/ml) in MM, in triplicate
    2. Add 1 ml of fresh media halfway through experiment
    3. Grow 7-8 days and quantify on multisizer
  4. 6 wells of a 6-well adherent plate for colony/suspension colony outgrowth:
    1. 20,000-35,000 cells per well (10K-17.5K/ml) in MC
    2. Add 1 ml of fresh media halfway through experiment
    3. Assay colonies at 7-8 days post plating
    4. Can collect suspension spheres and assay at the same time
  5. Flow cytometry

Use 50-100K per condition, use for EpCAM/CD49f/CD10 flow

Can use depleted fraction for single stains if necessary

  1. Cytospin

20K per spot

Ideally 4 spots per fraction; need 80K per fraction in 800 µl RMFC

12 slides per patient (24 total)

  1. Snap freeze pellets

Any leftover cells in significant quantity, pellet and snap freeze for RNA

Most likely depleted and EpCAM+ fractions

Medias, Buffers, etc.

 

Collagen Coating

Collagen stock at 4 mg/ml (check this, not always 4 mg/ml), dilute to 10 mg/ml (1:400 dilution)

Prepare enough to coat x number of wells with 0.5 ml

Incubate 1 hour at RT

Wash wells with 2×0.5 ml PBS

 

Matrigel Coating

Thaw matrigel on ice, using cooled pipet tips, pipet up and down to mix

Dilute matrigel 1:3 with MC (serum free)

Prepare enough matrigel to coat x wells with 300 ml

Incubate at RT 1 hour

Aspirate unbound matrigel and wash wells with 1×0.5 ml MC

 

MC (Mammary complete media = MEGM (MEBM + Bullet kit))

Purchased from Lonza (Cat# CC-3150)

MEBM basal media +

Bullet kit: Insulin (5 µg/ml), Hydrocortisone (0.5 µg/ml), hEGF (10 ng/ml), Pituitary Extract (52 µg/ml), Gentamycin (optional)

1% Antibiotic/Antimycotic (Invitrogen)

MM media (MEGM – pituitary extract)

Make as above for MC except leave out pituitary extract

Use for growing mammosphres.The pituitary extract precipitates gunk in the media that serves as scaffolds for cellular aggregation in non-adherent culture.  Note that I have tried but do not use the B27 supplement recommended in the Dontu Genes&Dev paper.  I found it to be unnecessary and that it made my cells more likely to aggregate.

 

Organoid Media (OM)

DMEM/F12 1:1 formulation (Invitrogen)

5% calf serum

Insulin (5 µg/ml), Hydrocortisone (0.5 µg/ml), hEGF (10 ng/ml)

1% Antibiotic/Antimycotic (Invitrogen)

RMFC (reduction mammary fibroblast complete media)

DMEM high glucose

10% calf serum

1% Antibiotic/Antimycotic

 

B1-can make ahead, store at 4C

This is my shorthand for buffer 1 for the Dynabeads.  This buffer is used to wash the beads and remove EDTA in preparation for digesting the DNA linker with DNase I.

 

PBS + 0.1% BSA

5 ml 1% BSA

            45 ml PBS

 

B2-can make ahead, store at 4C

This is my shorthand for buffer 2 for the Dynabeads, it contains EDTA to block endogenous DNases that might prematurely sever the bead connection to the IgG antibody. Resuspend cells in this buffer when adding beads.

 

PBS + 0.1% BSA + 2 mM EDTA

5 ml 1% BSA

            1 ml 0.1 M EDTA

            44 ml PBS

 

B3-make fresh, warm to 37C for bead release steps

This is my shorthand for buffer 3 for the Dynabeads, it is used during the DNase step when releasing cells from beads. It contains calcium and magnesium to chelate any EDTA left that might prevent DNase I from working. I am not sure how necessary it is to use RPMI media but I always have.

 

RPMI + 1% serum, 1 mM CaCl2, 4 mM MgCl2

0.5 ml FBS

1 ml 200 mM MgCl2

                0.5 ml 100 mM CaCl2

                48 ml RPMI

 

Notes from Patty on this protocol

I have adapted this protocol from the instructions with the Dynabeads.  The major changes involve releasing the beads with the DNase step, the instructions with the Dynabeads do not work

The dynabeads used are the CELLection Pan-Mouse IgG Dynabeads (Invitrogen 115-31D).  These are pre-conjugated to anti-mouse IgG secondary antibodies with a DNA tether.  Addition of DNase digests this tether, releasing the beads from the cells.  Note that the antibody complex (primary antibody and anti-mouse IgG) will remain bound to the cells post release (this a factor if you need to do any additional staining that requires anti-mouse secondary antibodies).

The advantage over beads from Stem Cell Technologies catalog #162-03.

You will need the Dynal magnet and all bindings need to be done in 15 ml conical tubes (also Invitrogen: DynaMag-15 cat# 123-01D).  When I initially prepare the beads, I bind the antibody to the beads in 1.7 ml tubes, rotating the tubes end over end and transfer them to a conical tube for washes.

I supplement the DNase provided with the Pan-Mouse IgG beads with DNase I from Roche, prepared at 5 mg/ml stock solution.

I generally get good viability post sorting but do expect significant die-off in culture (typically I get about half of what I thought I plated if I count the next day). Culturing primary cells can be tricky. I typically plate cells in adherent culture in what I call MC (see below) media and allow them to grow undisturbed (supplementing with media) for about a week before I decide to pool them or manipulate them in any way with Trypsin.  Usually many cells remain non-adherent and grow in suspension.  Those that do adhere, do so after a few days (1-3) and multicellular colonies form after 3-5 days, expanding more rapidly after 5 days or so.  Grow primary human cells in defined serum-free media to avoid enriching stray fibroblasts that invariably contaminate your cultures.

For mammosphere growths, we use the corning ultra-low attachment plates, they come in several sizes from 10 cm down to 24- or 96-well plates.  Corning catalog#s:  3262, 10-cm dishes; 3261, 6-cm dishes; 3471, 6-well; 3473, 24-well; 3474, 96-well.  I typically grow spheres at a concentration of 20,000 cells per ml (as per the Dontu Genes&Dev paper) for 7-10 days, supplementing with fresh media, but not changing the media every 3-4 days.