Reagents for 6.1

Reagents for 6.1 kdorfman Tue, 10/11/2011 - 22:38

If you used 10 mM Fe-citrate, instead of 89.4 mM, the calculations would be easier.

The 89.4 value is because Tobias Baskin's lab makes a 89.4 µM Fe-citrate medium, and it's easy to mix up a batch from the 1000x stock solution.

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

Each group does 4 coverslips, all of the same cell type. Some will use LLCPk, others will use 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 for coverslips 1-3

    • 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

  • high-Fe-HBS for coverslip 4

    • 2 mL aliquots COLD
    • 2 mL aliquots WARM
    • need some to make high-Fe-HBS-BSA for transferrin
    • Make 50 mL
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

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

    • 5 mg BSA in 5 mL High-Fe-HBS

  • Transferrin in Fe-HBS-BSA to treat coverslips 1-3.

    • 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
  • Transferrin in high-Fe-HBS-BSA
    • Each group does 1 50 µL treatment.
    • Make 0.44 mL: 7 µL AF-488-Tf + 0.433 mL high-Fe-HBS-BSA
    • Aliquot 55 µL
# aliquots 1 2 3 4 5 6 7 8
vi (Tf) 0.88 1.76 2.64 3.52 4.4 5.28 6.16 7.04 µL
HBS 54.1 108.2 162.4 216.5 270.6 324.7 378.8 433.0 µL
vf 55 110 165 220 275 330 385 440 µL

  • non-CO2 medium ICE COLD USE Fe-HBS - change manual !!!

    • 3 rinses per coverslip
    • 15 mL aliquots in the refrigerator
    • need over 100 mL

  • HBS-paraformaldehyde 3.7%

    • Need 60 mL
    • Divided in 2 aliquots, one per fume hood
      Ingredient units 10 15 20 25 40 50 60
      paraformaldehyde g 0.37 .555 2 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