5.1 2013

Submitted by kdorfman on Thu, 09/26/2013 - 14:45

W 10/23

Receptor-mediated endocytosis

NO LL's

NO high iron

3 coverslips of 3t3 per group

Try Lysotracker (Invitrogen L-7528)

  • 50 - 75 nM

  • stock = 1 mM in DMSO

  • 30 min - 2 hours incubation warm.

  • Add lysotracker to cells at 1 pm.

    • Make 100 µM stock
    • 1 µL of 100 µM stock to each dish. Assume ~1.5 mL per dish

Omit the high-iron treatment

For convenience, all staining, incubation, etc. will be done in HBS.

Each group does 3 coverslips of 3t3.

  • HBS to mix everything else in. There is a bottle of sterile 10X on the prep room bench.

    • 6 mL aliquots COLD (final rinse before fix coverslips 1-3)
    • 15 mL aliquots RT in hood (to rinse off fixative)
    • Need ~300 mL total

  • Fe-HBS

    • 20 mL aliquots COLD to rinse off growth medium, then tf
    • 5 mL aliquots WARM (incubation after tf treatment)
    • need some to make Fe-HBS-BSA for transferrin
    • make 200 mL
Solution stock conc final conc 25 50 75 100 200 mL
10X HBS 10 1 2.5 5 7.5 10 20 mL
Fe-citrate (mM) 89.4 0.1 27.96 55.92 84 112 224 µL

plus water to final volume

  • Fe-HBS-BSA 1 mg/mL to make transferrin solution

    • 5 mg BSA in 5 mL Fe-HBS

  • Transferrin in Fe-HBS-BSA

    • Alexa-Fluor 488 transferrin (Invitrogen T13342, MW = ~80KD) stock is 5 mg/ml (=62.5 µM). Final concentration = 1 µL.
    • Each group does 3 50-µL treatments (need extra for pipetting error)
    • Make 1.2 mL: 19.2 µL AF-488-Tf + 1.1808 mL Fe-HBS-BSA
    • Aliquot 165 µL
      if new transferrin stock is needed add 1 mL sterile distilled water to the new bottle.
# aliquots 3 6 9 12 15 18 21 24
vi (Tf) 2.64 5.28 7.92 10.56 13.2 15.84 18.48 21.12 µL
HBS 162.4 324.7 487.1 649.4 811.8 974.2 1136.5 1298.9 µL
vf 165 330 495 660 825 990 1155 1320 µL

Old fashioned way:

Ingredient units 10 15 20 25 40 50 60
paraformaldehyde g 0.37 .555 0.74 0.925 1.48 1.85 2.22
water mL 9 13.5 18 22.5 36 45 57.78
10X HBS mL 1 1.5 2 2.5 4 5 6

heat paraformaldehyde in water, then add 10X HBS
Add a drop of 10N NaOH
Heat and stir in the fume hood

OMIT HIGH IRON

Solution stock conc final conc 10 15 20 25 30 50 mL
10X HBS 10 1 1 1.5 2 2.5 3 5 mL
Fe-citrate (mM) 89.4 2 223.7 335.6 448 559.2 671 1118.4 µL

plus water to final volume