Horizontal Spread Assay

Horizontal Spread Assay

(on 10T1/2 mouse fibroblasts)

10T1/2 fibroblasts work well for this assay because they grow well in DME 10% IFS, are infectable with high efficiency, and can be killed off rather quickly with standard drugs.  Also, for routine passaging, the cells can also be split sparsely (a 1:10 or 1:15 split is usually fine) and they can sit at confluency for several days.

¨ Add ~ 2.5 x 105 cells per 10 cm plate at least 8 hr prior to infection.

¨ Infect cells for at least 4 hr with the supernatant from the tester cell line with 5µg/ml polybrene. For supernatants from HMECs or other cells growing in serum-free media, add DME 10% IFS so that the final serum concentration is ~2% on the 10T1/2 cells.

¨ Replace the media with DME 10% IFS and let the cells recover and grow for at least 12 hour and then add the following drugs.

Drug

Concentration

100% death on control plate

Neo 1 mg/ml Day 3 (from addition of drug)
Hygro 300 µg/ml Day 4 *
Puro 2 µg/ml Day 2
Zeo 1.2 mg/ml Day 6 *
Blast 15 µg/ml Day 3

* These plates will probably get confluent and the cells won’t die completely. They’ll probably need to be split ~ 1:4 on Day 3 in the presence of drug to see 100% killing. The cells will take a long time to be killed with Zeo, but resistant cells are smaller and healthier while sensitive cells flatten out.

Horizontal Transfer Test (PJ Keller additions)

Document this in the HT test lab notebook

Do always for retrovirus, can spot check for lentivirus or do for high titer preps.

 

After you have infected your cells of interest do one of the following:

A:  Selectable viral constructs (puromycin etc.)

Split infected cells into selection media and select colonies that have been infected by virus.

Post-selection, change the media to fresh media with out puro etc. and allow the cells to condition the media for at least 24 hours.  Collect this media, filter it to remove any cells and apply it, with polybrene/protamine sulfate, to a test cell line such as 293T etc.

Allow cells to infect as usual and then split into selection and monitor until all cells die (if a colony survives, your virus is replication competent and has failed the HT test)

B:  Non-selectable viral constructs with GFP marker (etc.)

 

Verify infected cells are green (etc.), usually this happens when titering virus or you can also check on the scope. Wash infected cells several times to remove any residual virus

Split infected cells onto a fresh plate and allow to condition media for at least 24 hours.

Collect the media, filter it to remove any cells and apply it, with polybrene/protamine sulfate to a test line such as 293T.

Allow cells to infect as usual and then verify the lack of GFP+ cells by flow cytomentry (preferred) or visual inspection (in this case, green cells mean that a replication competent virus was made and you failed the HT test).

Human 3D Collagen Morphogenesis Assay

1) Freshly dissociate organoids into single cells and then seed 500K in 500µl/well in a 48 well non-adherent plate with protamine sulfate with virus at MOI 3 for infection overnight.  (Patty and I have both stopped spin infecting the cells. This method seems to produce less cell death. I need to infect roughly 2x the number of cells that I will need for plating the next day to have enough for my experiments.)

2) The next day the cells are clumpy after infection so wash the cells in 5%CS DMEM and pass them through a 200µl pipette repeatedly until achieving a single cell suspension again.

3) Once acquired a single cell suspension, seed cells into either non-adherent plates for mammospheres or adherent plates for colony forming assays at a density of 20K/ml as described in Dontu et al. (When we plated dissociated cells from tumors, we plated at 5K/ml, thinking that more cells would form tumorspheres.)

The number of mammospheres that form following infection (or as freshly seeded cells) varies quite a bit among different patients.  Some patients form a ton of colonies, no suspension spheres, and medium numbers of mammospheres, etc.

4) Wait 7 days for mammosphere formation as described in Dontu et al.  (They don’t seem to grow larger (by eye) if you wait longer than that)

5) After 7 days, seed spheres  for the collagen gel experiments. Seed in the range of 1000 mammospheres/well in a 4-well chamber slide.  (To do this, multisize 3 wells of the 48 well plate, and plate 2-3 wells depending on the number that is calculate by multisizing).  Since replicates are similar for multisizing, this method seems to work well. 

The number of colonies that grow out of the mammospheres that are seeded are also patient sample dependent. The range is around 30-50% of mammospheres form some kind of colony. It seems that the patients that form the highest number of mammospheres have the lowest % of colony growth after seeding. By looking at the wells after seeding after each day you can see specifically which mammospheres are going to grow or not.

PGE2 Assay Protocol

Prostaglandin E2 EIA Kit Protocol
Adapted by Jenny 9/5/07

 

Date of CM pool and thaw: ____________

Date of assay: _____________

CAF/RMF line tested: ________________

DAY 1

  • Stock Solution Preparation
    • Use within 2 months of reconstitution
    • Store at 4 °C
    • USE ULTRAPURE WATER AS A SOLVENT UNLESS OTHERWISE STATED
    • For EIA Buffer Preparation:
      • Dilute the vial of EIA buffer concentrate with 90 mL of ultrapure water
    • For Wash Buffer Preparation:
      • Dilute the 5 mL vial of wash buffer concentrate with 2 L of ultrapure water and add 1 mL of tween 20 (i.e. dilute the wash buffer 1:400 and add tween 20 at 0.5 mL//L soln)
  • Standard Solution Preparation
    • Use the standard stock soln within 4 weeks of reconstitution, stored at 4 °C
    • Use all further diluted standard solns within 24 hrs
    • For the 10 ng/mL PGE2 stock standard:
      • Add 1 mL EIA buffer to the PGE2 standard vial
    • For the remaining serial dilution PGE2 standards:
      • *Use EIA buffer to further dilute UNLESS the samples will be diluted 1:10 in their respective culture medium; if this is the case, dilute the remaining standards in this culture medium diluent instead of EIA buffer

        Standard

        Formulation

        Final Concentration

        1 900 µl diluent* + 100 µl stock standard 1 ng/mL
        2 500 µl diluent* + 500 µl standard 1
        3 500 µl diluent* + 500 µl standard 2
        4 500 µl diluent* + 500 µl standard 3
        5 500 µl diluent* + 500 µl standard 4
        6 500 µl diluent* + 500 µl standard 5
        7 500 µl diluent* + 500 µl standard 6
        8 500 µl diluent* + 500 µl standard 7
  • Prostaglandin E2 AChE Tracer Solution Preparation
    • Use within 2 weeks of reconstitution and store 4 °C
    • Reconstitute the 100 dtn PGE2 tracer with 6 ml EIA buffer
      • Add 60 µl tracer dye soln to 6 ml soln
  • Prostaglandin E2 Monoclonal Ab Solution Preparation
    • Use within 4 weeks of reconstitution and store 4 °C
    • Reconstitute the 100 dtn soln with 6 ml EIA buffer
      • Add 60 µl antiserum dye soln to 6 ml soln
  • Strip Plate Set Up
    • Place unused strips at 4 °C after use; be sure the packet is sealed with the desiccant inside
    • Use the suggested plate format; label appropriately
    • The minimum for a decent analysis:
      • 2 blanks (Blks)
      • 2 non specific binding wells (NSBs)
      • 3 maximum binding wells (B0s)
      • an eight point standard curve done in duplicate
    • To start the assay, pipet the above reconstituted reagents according to the table (volumes are in ul), and incubate 18 hrs 4 °C with plastic film cover:

      Well

      EIA Buffer

      Standard or Sample

      Tracer

      Antibody

      Blk 0 0 0 0
      TA 0 0 5 µl, day 2 0
      NSB *100 0 50 0
      B0 *50 0 50 50
      Standard or Sample 0 50 50 50

      * if culture medium was used as the diluent instead of EIA buffer, add 50 µl culture medium to NSB and B0 wells, and 50 µl EIA buffer to NSB wells.

DAY 2

  • Developing the Plate
    • Reconstitute 1 dtn vial of Ellman’s Reagent with 20 ml of ultrapure water.
    • This reconstituted soln is unstable and should be used the same day as it is prepared; protect from light.
    • Empty the wells and rinse 5x with wash buffer
    • Add 200 µl Ellman’s Reagent to each well and 5 µl of tracer to the TA wells
    • Cover the plate and incubate in the dark, shaking, for 60-90 min.

PGE2 Assay Protocol ( Click to Download )

Primary Tissue Isolation-Freeze Down

Freeze Media

9ml DMEM + 10% CS + P/S/F (antibiotics/anti fungals)

1ml DMSO

  1. Remove tissues and place in sterile Petri dish.
  2. Chop/mince tissue with STERILE razor blade into 1mm cubes.
  3. Place 4-5 tissue fragments into a cryovial containing 1ml of Freeze Media.
  4. Place cryovial in Nalgene cell freezer and put at –80oC until next day. (If you do not use a Nalgene cell freezer, this protocol will NOT work)
  5. Transfer the cryovials into liquid nitrogen until use.

To thaw- place frozen cryovial in to 37oC water bath for 2-3 minutes.  Using sterile forceps remove tissue fragments and rinse in sterile PBS. Tissues are now ready for use or implantation.

Priming Cells with CAF or RMF CM

Priming Cells with CAF or RMF CM ( Click to Download )

ALL EXPERIMENTS INVOLVING PRIMING OF MCF7 CELLS WITH CAF OR RMF CM
  • Primed cells can be further analyzed by qPCR/WB, sphere formation, and in vivo tumor formation
  • To control for differences in CAF and RMF passage number, and any differences that might ensure because of this, it is best to seed a large number of cells for priming at the beginning and then disperse among assays:

PrimingWithCAF

To Prime Cells:

  1. Seed appropriate number (if using the experimental setup described above, seed at least 106) of MCF7 (preferably low passage) cells to either 10 cm plates (for 106 cells) or 6 well plates (for lower dilutions) in their normal growth media (DMEM + 10% CS + AB/AM).
  2. The next day, thaw CM from -80 freezer along with an aliquot of charcoal/dextran stripped FBS (CD-FBS) and 200 mM L-glutamine. Thaw only what you need- do not freeze/thaw CM.
  3. After 24 hrs, wash cells twice with PBS.
  4. Change appropriate wells to supplemented CM.
    1. Add back 5% CD-FBS to thawed CM aliquots.
    2. Add back 2 mM L-glutamine to thawed CM aliquots.
    3. Spike in any other treatments, such as an anti-IL6 blocking antibody or vehicle control.
    4. For wells receiving regular media (not CM), use PRF-DMEM + 5% CD-FBS + 2 mM L-glutamine + AB/AM with any additional treatments, such as 0.5 uM PGE2 or vehicle control.
  5. Take pictures after 24 hrs. Cells should look EMT’d in most conditions.
  6. Supplement with fresh media on day 3.
  7. Prepare for flow cytometry on day 6 (see protocol Flow Cytometry for CD44+/CD24-/EpCAM+ cells), RNA isolation (use Qiagen kit), or western blot (for RIPA extraction).