Rather than using citrate buffer, you can use 1x10-4N HCl, which will have a pH of ~ 4. STZ dissolves at this pH quite readily. The advantage of using HCl is that it has no buffering capacity and consequently very little possibility of systemic alteration in pH even when you use as much as 4ml/kg. But it is better to stay at or below 2ml/kg.

The key factors are to ensure that the STZ is stored properly before using the powder (dry at -20ºC or -80ºC), allowed to come to room temp before opening the bottle, weighed, dissolved and injected as quickly as possible. If all precautions are taken, virtually all rats will become diabetic and there should be no deaths!

After injecting STZ, I usually give about 1hr before rats are given food (and sugar water, when deemed necessary). There is no need to give sugar water beyond overnight   when STZ treatment is performed in the evening. By the next morning rats will already be diabetic. Sugar water is only necessary for the early period when there may be an excess of insulin released from damaged beta cells.

At first glance on publications concerning STZ its not so easy to induce diabetes with single dose! Especially your dose of 65mg/kg. Try to increase the dose or to inject two times. Here is just one title that I have looked and there are questions on RG that you can read concerning STZ and diabetes. 

Effect of Varying Dose and Administration of Streptozotocin on Blood Sugar in Male CD1 Mice

As for the fact that glucose is causing the reverse of STZ effects i dont think its possible.

all best

i agree,stz induces diabetes 1 and distroyed the islands, givIng glucose could not restore this fenotype

 I agree with Nicokola there is no way glucose can reverse STZ effects.

Few point that you may consider while injecting STZ:

Single dose not always helpful

Solution should be prepared fresh and injected as soon as possible till then on ice.

Fasting animals for 4-6 hr do help in better induction of diabetes with STZ

Good luck!

 Alex,

There are multiple factors that can influence your ability to destroy beta cells by STZ, including strain-dependent variations. 

In case of fairly common type of lab rats (not the people who work with them and also lovingly called lab rats), such as Lewis or Wistar, 65mg/kg dose of STZ when used properly, will cause partial destruction of beta cells. You should observe hyperglycemia but not as high as when you use higher dose. At 80mg/kg STZ, rats become reliably ragingly diabetic within 24hr-post treatment.

While others are correct that you cannot expect re-emergence of beta cells after they are completely destroyed, however, you have to be mindful that STZ uses Glut-2 (high Km, ~16mM) to gain entry into beta cells just as does glucose. In that context, you have to consider the possibility that by giving 20% glucose you may have sufficiently swamped the transporter by a large excess of a competing glucose molecule and considerably reduced the effective concentration of STZ that entered beta cells. If you fasted your rats before treating them with STZ and waited ~1hr before giving them glucose water, it is less likely that glucose had much of an effect on action of STZ.

Although there may be some reduction in beta cell mass in your case through partial destruction in your rats but not enough to cause overt hyperglycemia. I believe, this is probably what has happened and it can be confirmed by glucose tolerance test; you should see a slower dissipation of bolus glucose compared to normal rats. 

There are multiple threads on this topic at RG, you ought to search and read them to learn more about details to reliably get diabetic rats by using STZ. It is not a complicated method but it is finicky. In our hands, it always works but we have been using it for well over 2 decades. 

It is not possible to reverse glucose level after STZ administration due to destruction of B- cells. However, I think you might have  problem with the injection of STZ itself. ot STZ stability

Ali:

"One more thing, it seems that theoretically STZ diabetes develops after 72h of injection,"

I am not familiar with this theory, please elaborate.

One day of sucrose feeding after single dose of stz administration in absolutely crucial. The reason is pretty simple and straight forward. Stz has high toxicity towards beta cells, very specific too due to its entry through glut receptor of beta cells. Once inside, it causes DNA damage and NAD+ depletion, resulting in rapid necrotic cell death. This is associated with release of massive amounts of pre formed insulin, which could cause hypoglycemic death. Hence one day of sucrose. 

Insulin is rapidly cleared therefore continued administration of sucrose will not be necessary. 

The effect of single dose of stz is absolute. I speak from my experience with wistar rats. All u have to ensure is stz is dissolved in citrate buffer and administered within couple of minutes of solubilization. I recommend to weight convenient aliquots in micro fuge tubes, carry the dry powder on -20 ice packs to where animals are housed, dissolve on site and use in a minute. If u pre weigh and mark your animals, it should be possible to use up an aliquot of dissolved stz in a couple of minutes. Helps if citrate buffer is as cold as possible. 

 No it will not revers the functions of STZ action in rats. But generally 20% glucose will be administrated after 6 hrs of STZ post administration. Since STZ massively destruct the pancreatic beta cells so insulin levels will increase inside the cell which results in hypoglycemia. In order to overcome this glucose will be administrated after  after 6 hrs of STZ post administration. 

STZ is very unstable substance. It pH sensitive and quickly hydrolized in solution. I used single dose of 90 mg/kg for 21-day old rats in 100 Mmol citrate acid buffer with pH 4.2 - 4.3. pH was adjusted by NaOH. Solution was made just before injection. Result was about 75%. Rats were weaned one day before injection.

Also I know person, who works with adult rats (200 g). He used dose of 60 mg/kg in same citrate buffer. Result was proximal to 60%.

Whan i tried to use 1 Mol buffer rats had died during 15 minutes after injection.

"Whan i tried to use 1 Mol buffer rats had died during 15 minutes after injection."

Could you tell us why would you try 1M Citrate buffer at ~pH 4 and how much volume was injected in what size rats?

First of all, no 20% glucose will not reverse the effects of STZ. 

Second, I use STZ at 60 mg/kg i.v. in saline, not in citrate buffer, and get 100% efficacy. The key details in making it work are these:

1. Prepare the STZ immediately before injection (

thank you all

We have prepared citrate buffer ice cold pH 4.5, STZ 65mg/kg, injected immediately after preparation, protected from light on 10/4/2015, . will be checking the glucose level

Could you tell us why would you try 1M Citrate buffer at ~pH 4 and how much volume was injected in what size rats?

I tired various concentrations of citrate buffer, that i had found in literature. The hifhest was 1 M. Volume was 1 ml per rat. Rats were 21-day old, so their weighr was proximal to 30 g.

Viacheslav,

A rat weighing 30g will have ~2.4ml blood volume. Blood does have a limited buffering capacity but not enough for such a challenge as you put them through.

When you dilute ~2.4ml blood volume with 1ml of 1M citrate buffer at pH ~4, you end up with ~0.3M Citrate buffer which will be perfectly capable to maintaining a pH close to the original - around 4; at the most, it may go up ~5. Death is a completely predictable consequence of your experiment, which could and should have been avoided by putting a little thought in the process.

In most cases, institutional IACUC will not (and should not) permit such experiments without a very strong justification.

Agree with Dr. Alam. I strongly insist on a volume of 1ml per kg. Its convenient too. Of course, there have been situations where I went up to 2 ml per kg bw when I couldn't dissolve substance s conveniently to be administered at a vol if 1 ml per kg.. One ml per a rat of 30g is tooooo much. 

After induction of STZ when we can give "glucose water" and "food" ?

What is the ideal body weight of rats?

24 hrs of glucose water is more than sufficient. I never fasted my rats before stz. In case of fasting, both can be started 15 mins after stz, I suppose. 

Rather than using citrate buffer, you can use 1x10-4N HCl, which will have a pH of ~ 4. STZ dissolves at this pH quite readily. The advantage of using HCl is that it has no buffering capacity and consequently very little possibility of systemic alteration in pH even when you use as much as 4ml/kg. But it is better to stay at or below 2ml/kg.

The key factors are to ensure that the STZ is stored properly before using the powder (dry at -20ºC or -80ºC), allowed to come to room temp before opening the bottle, weighed, dissolved and injected as quickly as possible. If all precautions are taken, virtually all rats will become diabetic and there should be no deaths!

After injecting STZ, I usually give about 1hr before rats are given food (and sugar water, when deemed necessary). There is no need to give sugar water beyond overnight   when STZ treatment is performed in the evening. By the next morning rats will already be diabetic. Sugar water is only necessary for the early period when there may be an excess of insulin released from damaged beta cells.