Culturing HMEC, vHMEC and HME lines (PJ Keller)

Culturing HMECs ( Click to Download )

HME/C lines are slower to trypsinize than most cells. Use care when passaging to avoid over-trypsinizing and damaging cells.

 

  1. Grow cells to 70-80% confluence in MEGM media.
  2. Aspirate media from the plate; rinse with 10 ml PBS to remove cellular debris and traces of serum (though most HMEC lines are grown in serum-free conditions, this step is still done to wash the cells).
  3. Pipet 2 ml of trysin-EDTA  (we use 0.05% Trypsin) onto the cells in the dish (full coverage is essential for trypsinizing the HMECs, use up to 3 ml for 10 cm plate).  Incubate at 37oC for 6 minutes to dislodge most cells. Firmly tap the plate on the side of your hand to assist dislodging. Pipet the trypsin forcefully over the cells several times with a p1000 pipet tip to further dislodge any stubbornly stuck cells. If cells are still not dislodging do not look balled up at all under the microscope, return the cells to the incubator for 1-2 minutes more and repeat.
  4. For a 10 cm plate, pipet 8-10 ml of OM media onto the plate while tilting the plate.  Rinse the cells off the dish moving the pipet from the top to the bottom of the tilted plate.  Repeat several times.  Transfer cells to a 15 ml conical tube.
    1. If passaging cells for growth curves, it is useful to remove the cells from the plate with 5 mls and then rinse the plate with an additional 5 mls of media to make sure all cells are collected for counting.
  5. Pellet cells at 1000-1200 rpm for 5 minutes.
  6. Prepare the plate(s) you are passaging the cells onto by labeling them with the new passage number (P1, P2 etc.). Pipet 9 ml of MEGM media onto each plate.
  7. Resuspend the pellet in the amount of growth media that corresponds to the ‘split’ you are doing.  For a 1:4 split, for example, resuspend the cells in 4 mls of media. Pipet up and down until the pellet disappears; transfer 1 ml of the cells to the prepared plate(s).  Swirl and rock the plates to evenly distribute the cells and place in the incubator. In general, HMECs should be split 1:3 to 1:5, about 1X per week.
    1. If you are passaging the cells for growth curves, resuspend the cells in 2 mls of MEGM and count. Plate a defined number of cells for your passage. To calculate population doublings use the formula: PD = log(A/B)/log2 where A is the cells collected and B is the number of cells plated initially (Holst, CR et al. 2003 Cancer Research)
  8. To freeze cells, follow protocol through step 5. Resuspend at 1-2 million cells per ml in freezing media (OM media + 10 % DMSO and extra 5-10% CS). Aliquot 1 ml per vial and freeze in isopropanol freezing container at -80oC for up to 3 weeks. Transfer to liquid nitrogen for long term storage.

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)

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)

DMEMF12 Mammary Epithelial Growth Media

DME/F12 – Mammary Epithelial Growth Media  11/00

 

Make a 1:1 mixture of DME and F12 media

Add these supplements to a 500 ml bottle of DME/F12; 1 tube of each.

 

 

Final conc.

1. hEGF 0.5 ml of 10 µg/ml 10 ng/ml
2. Hydrocortisone 0.5 ml of 0.5 mg/ml 0.5 µg/ml
3. Insulin 1 ml of 5 mg/ml 10 µg/ml
4. Pen/Strep/Fungizone 5 ml  

Information on ordering and aliquoting the supplements:

hEGFSigma E-9644   0.2 mg  $125.50  enough for 40 bottles.  Dissolve in 20 ml 10mM Acetic Acid, 0.1% BSA make 500 µl aliquots of 10 µg/ml.

HydrocortisoneSigma H-0888 1g $16.10.  Dissolve in 95% Ethanol and make 500 µl aliquots.  100mg/200ml 95% Ethanol for a final conc. of 0.5 mg/ml.

InsulinSigma I-2767  100 mg $89.20 enough for 20 bottles.  Dissolve in 20 ml 0.005 N HCL and make 1ml aliquots 5 mg/ml. Conc. HCL is 11.6 N. Make 1 N HCL and then add 100 µl to 20 ml H2O.

Drug Stocks for Mammalian Cells

 

 

Stock concentration

Storage

1 Neomycin (G418) 500 mg/ml -20oC
2 Puromycin 2.0 mg/ml -20oC
3 Hygromycin 100 mg/ml 4oC T.C.
4 Zeocin 100 mg/ml -20oC
5 Blasticidin 10 mg/ml -20oC

Information on ordering and aliquoting :

Neomycin (G418, Geneticin)Life Tech. 11811-031  5g $265.90.  Comes lyophilized,  store at RT. Dissolve in PBS-. Usually, it’s activity is ~730µg/mg in which case, resuspend in 7.3 ml PBS for a 500 mg/ml stock.

PuromycinSigma P7255 100 mg $113.60.  Dissolve in 50 ml PBS- for a final conc. of 2 mg/ml.

HygromycinSigma H3274   1 g $263.50 comes lyophilized,  store at 4oC. Add 8 ml H2O and it rapidly goes into solution.  Bring volume to 10 ml for a final conc. of 100 mg/ml.  Make 500 µl aliquots and store at 4oC in dark.

ZeocinInvitrogen R250-05   5g  $725  Comes as a liquid at 100 mg/ml.

Blasticidin S HCLInvitrogen R210-01  50mg $135.  Dissolve in 5 ml H2O for a final conc. of 10mg/ml.

ELISA Protocol (J Rudnick)

Adapted by JAR 10/4/09

ELISA PROTOCOL ( Click to Download )

ELISA kit: cat # _____________ from EBioscience

Reagents:

Buffers:
1. Coating Buffer
1 packet ELISA coating buffer (cat # 00-0044-59)
1L DI water
filter using a 0.22 µm filter

2. Wash Buffer
1X PBS + 0.05% Tween

3. Assay Diluent
10 ml 5X assay diluent bottle (cat # 00-4202-43)
40 ml DI water

4. Capture Ab
48 µl of Capture Ab soln (cat # 14-7069-67)
12 ml coating buffer

5. Standard- rhIL6 at 1 µg/ml
To prepare 200 pg/ml soln, add 5 µl standard soln (cat # 39-8069-60) to 25 ml assay diluent
To prepare subsequent serial dilutions, use 500 µl of preceding standard soln and 500 µl assay diluent
*these are single use vials. Dilute as suggested above.

6. Detection Ab- Biotin conjugated anti-IL6
48 µl Detection Ab soln (cat # 13-7068-67)
12 ml assay diluent

7. Enzyme- Avidin HRP
48 µl Avidin HRP soln (cat # 18-4100-93)
12 ml assay diluent

8. Substrate Soln
Working dilution as prepared (cat # 00-4201-52)

9. Stop Soln
1 M H3PO4

Before Beginning ELISA

  1. Prepare Coating Buffer.
  2. Coat ELISA plate with 100 µl/well of capture antibody diluted in Coating Buffer.
  3. Seal the plate and incubate overnight 4oC.

Experimental Procedure

  1. Aspirate wells and wash 5X with 200 µl/well wash buffer. Allow time for soaking between washes (1-2 minutes).
  2. Blot the plate on a paper towel to remove any residual buffer.
  3. Block wells with 200 µl/well 1X Assay Diluent. Incubate 1 hr RT.
  4. Aspirate and wash as in step 1.
  5. Add 100 µl diluted standards to each well (standards set up in duplicate). Perform 2 fold serial dilutions of the standards to generate a standard curve. Add 100 µl diluted samples to the appropriate wells (samples set up in triplicate). Seal the plate and incubate O/N 4oC.
  6. Aspirate and wash as in step 1.
  7. Add 100 µl/well Detection Ab to all wells. Seal the plate and incubate RT 1 hr.
  8. Aspirate and wash as in step 1.
  9. Add 100 µl/well Avidin-HRP soln to all wells. Seal the plate and incubate RT 30 min.
  10. Aspirate and wash 7X with 200 µl/well wash buffer. Allow 2 min soaking time between washes.
  11. Add 100 µl/well Substrate soln to all wells. Seal the plate and incubate RT 15 min.
  12. Add 50 µl stop soln to all wells.
  13. Read the plate abs at 450 nm.

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.