Insect haemolymph cells can be isolated from Plodia interpunctellalarvae via a small bore glass capillary tube. Normally 2-3 ml can be isolated in this way. This primary isolate can be transferred to 200 ml PBS in a 5ml Falcon tube.
Absolute Cell Counting
The haemolymph of Plodia interpunctella larvae usually contain a total of 3,00-6,000 cells and counting these manually on a Haemocytometer is time consuming and not very accurate as counts estimated can be as low as zero. The use of flow cytometry to calculate the absolute count by adding latex beads of a known concentration to a 1:100 dilution of haemolymph cells allows counts to be determined in 30 seconds when counting 2,500 beads (20 ml volume) from a solution of 180 ml PBS. Normally the average rate of cell counting manually is over 5 mins, so large numbers of haemolymph cells can be counted in a relatively short time by flow cytometry.
Insect haemophyl cells viability can be assessed by propidium iodide (PI)
- Cell count protocol
- Make a dilution of latex beads in PBS.
- Count beads on a Haemocytometer.
- Add a known number of beads (20,000) accurately to a known volume of sample.
- Draw an event stop gate on bead population to stop at 2,000 events.
- Other gated events are a proportion of the bead count.
- Viability protocol
- Resuspend cells in 0.5 ml PBS.
- A) Add 5 mg/ml PI or B) Add 2.5 mg/ml 7-AAD or C) Add >200 ng/ml DAPI or D) Add 50 nM ToPro-3 Add 1ul per 100ul cells of DRAQ7 stock solution.
- Collect 10,000 events as soon as possible, using the required fluorescence channel, see below.
- PI – 575 nm, 610 nm or 670 nm channels from Argon laser.
- 7-AAD 670 nm channel from Argon laser.
- DAPI 440 nm channel from UV laser or Violet diode.
- ToPro-3 660 nm channel from Red HeNe (633 nm line).
- DRAQ7 695 nm or 760 nm channel from Red HeNe (633 nm line). DRAQ7 695 nm or 760 nm channel from Argon laser.
- Absolute cell counting formula