Category: Angular Scattering

Cell Media/Solutions

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Cell Media (EMT6)

  • 500 mL BME (Basal Medium Eagle. We have always gotten this from the Foster lab)
  • 2.5 mL Pen Strep
  • 2 mL Fungizone
  • 1 mL plasmocin
  • 1 mL primocin
  • 10% FBS – 55.5 mL (Fetal Bovine Serum)

*Note: 20% FBS media is also needed for when cells are first thawed.

*For SCC7 cell line, components are all the same except instead of BME base media, use RPMI media

Cell Media (HeLa)

  • 500 mL RPMI 1640
  • 10% FBS (50 mL)
  • 5 mL Hepes Buffer
  • 5 mL Pen Strep

*All Nada solutions are based on 500mL total final solution

Nada Solution #1 (basic cell measurement solution)

  • 3.9447 g NaCl
  • 0.2199 g KCl
  • 0.9008  g D-glucose
  • 6 mL Hepes
  • 0.0832 g CaCl2
  • 0.0571 g MgCl2

Nada Solution #2 (permeabilizing solution)

  • 3.9447 g NaCl
  • 0.2199 g KCl
  • 0.9008 g D-glucose
  • 6 mL Hepes
  • 0.476 g MgCl2
  • 20 μM Ionomycin

Nada Solution #3 (calcium shock solution)

  • 3.9447g NaCl
  • 0.2199g KCl
  • 0.9008g D-glucose
  • 6mL Hepes
  • 0.0888g CaCl2

SUPERFASLIGAND (for 100 ng/10 uL concentration)

  • 5 ug of SFL
  • 500 uL of HeLa Media

9/9/15 IRAM Meeting

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During the weekly angular scattering group meeting we discussed:

  • Incubator: Cells are never growing to confluence in the incubator. Yesterday frozen SCCVII cells were brought over to our lab from the Foster lab, thawed, and put into media. Today all of the cells were dead. How can we confirm that the incubator is the problem? If it is, then the most likely issue is the % humidity. There is usually condensation on the incubator door, and the water bath is evaporating quickly.
    • We will contact Phoenix Equipment about possible sanity checks/repairs or purchasing a new incubator.
  • Cell measurements will be on hold for about a week while the Foster lab gets CO2 for their incubator and gets new cells up and running.
  • Reinstating the holographic arm of the elastic system: Determining how much more stable size estimates we can get by going to lower angles and (a) doing no processing, (b) intensity smoothing, (c) coherent smoothing of speckle. Eventually move to a common path interferometer? ROBERT
    • We need to buy another BK7 flat or wedge so that we can put in a double bounce periscope that preserves polarization, like Zach’s system had.
  • Reinstating the Raman arm (probably not in the near future)
  • Measure the scattering from multiple (>5) small beads to see the effect of speckle on the fits when we know what the right answer should be. JANET
  • Possibility of larger field of view when doing interferometric measurements
  • Possible uses for SLM
  • Update on Xing’s sparse sampling simulation study. Xing is creating a sparse distribution of scatterers (mitochondria) and converting the sparse probability density function (versus diameter) into a cumulative distribution function. This CDF is then being fit, and parameters like the maximum diameter (dmax) and diameter that equally divides the area under the PDF (d50) are reported. We are looking for robust parameters. This will tell us how much the sparseness of our sample alone is limiting the stabilty of our fits.
    • The next step is to repeat this process ~100 times. XING