Manduca Cell Solutions



MANDUCA HEMOCYTE BUFFERS - modified 8.27.98 in the 4x stocks to make final concentrations of 5mM PIPES; 6mM Calcium and 37 mM KCl

4x Buffered Salt Stock Solution: (1000 mls) for making MSB (Manduca Saline Buffer) and AC-saline
amount compound final concentration
0.8ml 5M NaCl 4mM
10.9g KCl 36.6mM
4g polyvinylpyrollidone (Sigma P-0930, 40,000MW)
10 ml 0.5M PIPES (blue tape, in Buffers) 5mM

Add 800ml dH2O in beaker, add PIPES and NaCl solutions and KCl and adjust to pH between 6.5-6.6 with 5N KOH. Then add polyvinyl pyrollidone. Adjust volume to 1000ml. Can autoclave to sterilize.

4x AC-citrate stock: (1000 mls) for making Anti-Coagulant Saline
  • 4 g citric acid monohydrate
  • 16 g sodium citrate (fw 294)
  • 6 g EDTA (mw 372) [or use 32ml 0.5M EDTA pH 8.0]
Add dH2O to less than 1000mls and pH to 6.5. Adjust vol to 1000ml and put in 500 ml bottles. Autoclave 20 minutes to sterilize. Store at room temperature.

Anti-Coagulant Buffer from 4x Stock: (1000 mls)
  • 250 ml 4x Buffered Salt stock
  • 250 ml 4x AC stock
  • 50 g sucrose
Add dH2O to less than 1000mls and pH to 6.5-6.6 with KOH and HCl as required. Adjust vol to 1000ml. Put in 100ml bottles. Autoclave to sterilize.

Manduca Saline Buffer from 4x Buffered Salt Stock: (100 mls)
MODIFIED 8.27.98 to increase Calcium to 6 mM. This doesn't store well. Can autoclave to sterilize, then it keeps well-but don't over-autoclave or the sugar will carmelize. For 100 ml bottles, 20 minutes autoclaving should suffice.
  • 25 ml 4x buffered salt stock
  • 1.8 ml 1M magnesium chloride (to make 50ml 1M MgCl2.6H2O, fw is 203.3 so 0.05x203.3=10.2g)
  • 0.6 ml 1M calcium chloride (so 6 mM)
  • 5g sucrose
check pH is around 6.5 (6.5-6.6). Then, autoclave to sterilize, then aliquot with sterile technique into 15 ml sterile tubes.

5% Formaldehyde Fixative: (10 mls) Be careful, formaldehyde is toxic!
  • 0.5 g paraformaldehyde
  • 10ml Manduca Saline Buffer no Ca2+ no Mg2+ + 2 drops 0.5M KOH. Heat in hot water bath (55-60C) to get into solution.
Check pH is around 6.4-7.0.

Manduca saline Plus BSA + 0.05% Tween20 to block slides:
To 10ml Manduca saline above add 0.2g BSA and 500µl 1% Tween20.

ORIGINAL SOLUTIONS
(from Tina Trenczek, then modified - instead of 83g sucrose per liter, better morphology if only 50g/li)

ANTI-COAGULANT SALINE SOLUTION (1liter)
amount compound final conc.
0.23g NaCl (fw 58.44) 4mM
2.98g KCl (fw 74.5) 40mM
2.978g EDTA (mw 372) 8mM
(16ml) (0.5M EDTA)
1.985g Citric acid-monohydrate 9.5mM
7.980g Na-citrate (fw 294) 27mM
50g sucrose
1g polyvinylpyrollidone
0.58g PIPES (fw 302.4) 1.7mM



Manduca SALINE BUFFER SOLUTION (Peter Cherbas)
amount compound final conc.
0.23g NaCl 4mM
2.98g KCl 40mM
3.66g MgCl2-6HOH 18mM
0.33g CaCl2 3mM
83g*** sucrose
1g polyvinylpyrollidone
0.58g PIPES 1.7mM
  1. Make PIPES, pH to 6.5 then add remaining, adjust to pH about 6.8 with 5N KOH. Adjust volume to 1 liter.
  2. For 5% paraformaldehyde fixing solution, leave out the MgCl2 and CaCl2. Though doesn't hurt to have it in.
  3. Ann Lackie used 305mOs; Cherbas' 412 mOs. Manduca hemolymph is about 300mOs.
  4. Manduca saline above doesn't keep well - tends to grow things. Instead, make 2.5x stock leaving out magnesium, calcium and sucrose - add these later. Alternatively, can make it, autoclave minimally (so doesn't carmelize), then portion to sterile tubes, discard after tube has been opened.
  5. ***Sucrose concentration is probably wrong for Manduca because hemolymph usually floats on the surface - using 50 instead of 83g per 100ml gives better morphology.
  6. If adding PTU (phenylthiourea - an anticoagulant) use 15mg/li. If you want good morphology don't add it.
  7. The solutions with sucrose in them will grow bacteria. In some cases one can add sodium azide to prevent growth - this may impair normal hemocyte functions like spreading (it certainly seems to). Or, filter sterilize or autoclave the solutions - do not over - autoclave or the sugar will carmelize.
  8. If want to add penicillin, add 30mg/liter.


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Last update Aug 1, 2000
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