HI Sarooj,

TM6B generally carries the Tb1 dominant mutation, which is responsible for the Tubby body (short body phenotype).

I can suggest two options for you.

1st option is easy way

If PTPmegKD/ + is not pupal lethal, you can collect non-tubbies at the pupal stage. It is easy to differentiate at the pupal stage on the 4th day of the cross.

2nd option has more steps

If you must get at the larval stage. I will suggest the following steps.

i) cross eight virgin females (PTPmegKD/TM6B) with four males ( WIII8 +/+ ) or vise verse in single food vial only for 2-3 hours.

ii) So that flies mate and lay eggs on food within a window period of 2-hour. most of the larvae at same instar stage. (if you dont narrow the window for egg lay, you may confused with 2nd instar for tubby larvae)

iii) Remove parental flies. Leave only eggs on vials at 25C incubator. On the third day, you may see third instar larvae.

iv) on the third day, add 35% glucose solution on food vial, and gently scrub the food with a brush. All the larvae started to float on glucose solution (due to solution density).

v) Pour the solution into a clean Petri dish and collect longer larvae (not tubby) by picking one by one with brush under the microscope into a new Petri dish with 1XPBS.

Note (keeping in PBS will help to get rid of glucose solution, if you don't wash larvae with PBS, larvae may be stuck and die due to stickiness of the glucose).

vi) Collected long larvae can be kept in separate new food vials for them to grow.

Good luck with experiments.

Jayasimman Rajendan thankyou so much for such detailed explanation.

The easiest way to do that is using TM6B, Tb1 balancer and sort the larvae with Tubby phenotype.

Otherwise you can use any of the long list of third chromosome balancer with GFP and analyze the larvae for green fluorescence.

https://bdsc.indiana.edu/stocks/balancers/balancer_chr3.html