Online Protocol

ONLINE PROTOCOL FOR THE Limnoperna fortunei GENOME PROJECT

07/05/2014

Limnoperna fortunei mussel that will be used DNA extraction and sequencing of the genome is registered under the accession number 19643 at the Malacologia Laboratory at the Biology Institute at the Federal University of Rio de Janeiro – Brazil.

 

13/04/2014

Quantification of the C content of L. fortunei hemocytes cells by flow cytometry technique.

This experiment was developed at Fio Cruz Institue, at Cardoso Fontes building, in partinership with Rubem Barreto at 05/12/13.

To determine the C content of L. fortunei cells we have used as main protocol the proposoal from the article by Lamatsch et al, 2000 (Cytometry 39:91–95) and the human C content as standard value from Tiersch et al., 1989 (Cytometry 10:706-710).

The L. fortunei mussels were collected from Jacuí lagoon at RS state and mantained in aerated water (12h light/12h dark) and feeded regularly. The specimens were transported to Fio Cruz 2 hours before experiment.

The haemocytes were  collected from 5 mussels with sterilized cold seringes, polled and mantained on ince. The standard human PBMC cells were collect through a ficoll gradient.

All samples were submitted to 6 minutes heat shock (60 celsius) and collored with PI (propidium iodide) 30ug/mL and then submitted to flow cytometer scan.

Results:

Nuclear DNA contents of target species were estimated in relation to an assigned value of 7.0 pg DNA for haploid male human cells (Tiersch et al. 1989) according to the formula, pgDNA= 7.0X/H, where X is the fluorescence channel number of the mussel sample and H the one of male human cells.

 

haemocytes

First peak represent non-collored cells for L. fortunei heamocytes and human PBMC in both graphs. The second peak are the PI-collored cells. The median values for L. fortunei haemocytes was 1187,2 and for Human PBMC 4357,3. If we take in consideration the 7pg valeu for haploid human cells and multiple by 2 (as PBMC are diploid cells), we have pgDNA=14*1187,2/4357,3. pgDNA= 3,82 pg in L. fortunei diploid haemocyte cells.

If we consider that diploid human genome has 6Gb base pairs, we conclude L. fortunei diploid genome to be 1,63Gb.

23/08/2013

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Counting cells.

OBS: I’ve estimate the number of hemocytes/ mL of hemolymph in order to determine the ideal number of cells to load at cytometer for DNA content estimation and karyotyping experiments.

For so, I’ve extract hemolymph from L. fortunei mussels, mixed with and antiplatelet, dye and counted at the Neubaeur chamber.

Calculation: The depth of the counting chamber is 0.1 mm3. If we want to find the number of cells per mL then we have to multiple by 10,000 as:

1 mL = 1cm3

1 cm3 = 1,000 mm3        

camara de neubauer

 

Then we have:

No of cells / mL =(total cells/

No of quadrants) * dilution factor * 10,000

 

 

 

First counting experiment 22/08/2013

1-      Syringe with 200 µl of MAS+formol on ice.

2-      Pool of hemolymph from three mussels ( ± 1,5 cm). Total volume: 350 µl + 200ul MAS+formol = 550 µl final volume.

3-      50 µl of final solution from step 2 + 10 µl of Giemsa.

Quadrants

No cells

upper left

47

upper right

36

bottom left

54

bottom right

23

4- 20 µl of solution from step 3 on Neubauer Chamber for counting. (To count the 4 external quadrants).

5-      Calculation

No of cells / mL =  (40*1.88*10000) = 752,000 or 7.5 x 105

 

Second counting experiment 23/08/2013

Two countings

1a- Syringe with 100 µl of MAS + forml on ice.

2a- Hemolymph from the biggest mussel we had (3 cm). Total volume: 300 µl + 100ul MAS+formol = 400 µl final volume.

3a- 50 µl of final solution from step 2 + 10 µl of Giemsa.

4a- 20 µl of solution from step 3 on Neubauer Chamber for counting.

Quadrants

No cells

upper left

16

upper right

26

bottom left

30

bottom right

29

5a- Calculation

No of cells / mL =  (25.25*1.56*10000) = 393,900 or 3.9 x 105

 

1b- Syringe with 100 µl of MAS + forml on ice.

2b- Pool of hemolymph from three mussels (± 1 cm). Total volume: 100 µl + 100 µl MAS+formol = 200 µl final volume.

3b- 50 µl of final solution from step 2 + 10 µl of Giemsa.

4b- 20 µl of solution from step 3 on Neubauer Chamber for counting.

Quadrants

No cells

upper left

51

upper right

27

bottom left

52

bottom right

29

 

5b- Calculation

No of cells / mL =  (39.75*2.4*10000) = 954,000 or 9,5 x 105

Considerations:

1- More than 5 million cells / mL can clog the cytometer. Our samples should be fine as we have less than that, but still a good amount to be detectable.

2- In the counting from sample B it was clear the predominant presence of agranular-small-size hemocytes. This sample was a pool of three very small mussels (1 cm). If size is related to stage of life, these were young mussels. So, the question is: does young mussels have populations of agranular small (immature?) cells that differentiate as mussels go to adulthood and face environmental challenges?