Cell -Freeze Down

/0 Comments/in , , /by

Freeze Media

9ml media the cells grow in

1ml DMSO

  1. Trypsinize as normally.
  2. Spin cells and resuspend in appropriate freeze media for ~1-2million cells/vial.
  3. Place 1ml of Freeze Media.
  4. Place cryovial in Nalgene cell freezer and put at -80°C 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.

Collecting CM From CAFs or RMFs (J Rudnick)

/0 Comments/in , , /by

Collecting CM From CAFs or RMFs ( Click to Download )

  1. Seed fibroblasts at one of the following cell densities depending on cell number: 100% confluence, 106 cells per 10 (dense) or 15 cm (sparse) plate, or 5×106 cells per 15 cm plate. Be consistent if comparing CM potencies among various patient fibroblasts. Try to match passage number if possible, although this is not always possible.
    1. I use PRF-DMEM + 2% CD-FBS + 2 mM L-glutamine + AB/AM for a 3 day CM collection (i.e. collect CM after 72 hrs).
    2. For CM analysis by WB or ELISA, it is better to use S/S CM. For this, use PRF-DMEM + 0.5% CD-FBS + 2 mM L-glutamine + AB/AM for a 24-48 hr CM collection (72 hrs and the cells start to look pretty miserable).
  2. If administering a treatment (such as PGE2 or EtOH vehicle control), add this at the time of seeding in PRF-DMEM conditions. I use PGE2 at 0.5 µM. I am able to still see a phenotype by only adding PGE2 1X (at start of experiment) over 72 hrs, but some say PGE2 is unstable; may want to supplement with fresh PGE2 every day and see if effect is more robust, if this is a concern.
  3. At 72 hrs, harvest CM from cells and filter using a Steriflip 0.22 µm filter. Aliquot CM at volumes that will be used for a particular assay. If CM is being used to prime MCF7 cells, typically 12 mL aliquots will suffice. Do not F/T and try to reuse for another assay.
  4. Store all CM at -80°C.

Crystal Violet Assay for Proliferation (APS)

/0 Comments/in , , /by

Crystal Violet Staining Protocol for Quantifying Proliferation

  1. Plate 25k cells in a 12 well plate or 50k in a 6-well plate
  2. Keep an empty well w/media to use as a blank
  3. Aspirate media, wash cells 1x with PBS
  4. Fix the cells with 10% buffered formalin for 30 min (use 1mL/well)
  5. Wash the cells 1x with ddH2O
  6. Add crystal violet solution and stain for 2 hrs
  7. Place cells on ice and rinse 3x with ddH2O
  8. Allow to dry for 5hrs-4wks
  9. Elute the dye with 10% glacial acetic acid (2ml) and read the OD at 600nm. Dilute and re-read over OD600 = 4.0.

Culturing HMEC, vHMEC and HME lines (PJ Keller)

/0 Comments/in , , /by

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

/0 Comments/in , , /by

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:

hEGF – Sigma 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.

Hydrocortisone – Sigma 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.

Insulin – Sigma 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

/0 Comments/in , , /by

 

 

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.

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

Hygromycin – Sigma 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.

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

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

ELISA Protocol (J Rudnick)

/0 Comments/in , , /by

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.